Note: Descriptions are shown in the official language in which they were submitted.
CA 02725026 2010-12-10
METHODS OF TREATING PLATINUM-SENSITIVE RECURRENT OVARIAN
CANCER WITH 4-IODO-3-NITROBENZAMIDE IN COMBINATION WITH AN
ANTI-METABOLITE AND A PLATINUM COMPOUND
RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Application No.
61/351,785,
filed June 4, 2010, the contents of which are hereby incorporated by reference
in its entirety.
BACKGROUND OF THE INVENTION
[0002] Cancer is a complex family of diseases affecting nearly every tissue in
the body
and characterized by aberrant control of cell growth. The annual incidence of
all cancer types
is estimated to be in excess of 1.3 million cases in the United States alone.
While a number
of first line therapies for the treatment of different types of cancer have
been deployed with
varying degrees of success, including surgical resection, radiation therapy,
chemotherapy,
and hormone therapy, it remains the second leading cause of death in the U.S.,
with an
estimated 560,000 Americans dying from cancer every year.
[0003] Malignant uterine neoplasms containing both carcinomatous and
sarcomatous
elements are designated in the World Health Organization (WHO) classification
of uterine
neoplasms as carcinosarcomas. An alternative designation is malignant mixed
Mullerian
tumor (MMMT). Carcinosarcomas also arise in the ovaries, Fallopian tubes,
cervix,
peritoneum, as well as in other non-gynecologic sites, but with a much lower
frequency than
in the uterus. These tumors are highly aggressive and have a poor prognosis.
Most uterine
carcinosarcomas are monoclonal, with the carcinomatous element being the key
element and
the sarcomatous component derived from the carcinoma or from a stem cell that
undergoes
divergent differentiation (i.e., metaplastic carcinomas). The sarcomatous
component is either
homologous (composed of tissues normally found in the uterus) or heterologous
(containing
tissues not normally found in the uterus, most commonly malignant cartilage or
skeletal
muscle).
[0004] Previous studies investigating a number of single agents in
carcinosarcoma of the
uterus have reported the following response rates: etoposide (6.5%);
doxorubicin (9.8%);
cisplatin (18%); ifosfamide (32.2%); paclitaxel (18.2%); and topotecan (10%).
Thus the
three most active agents discovered to date include cisplatin, ifosfamide, and
paclitaxel. A
randomized phase III trial comparing ifosfamide to ifosfamide plus cisplatin
showed an
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CA 02725026 2010-12-10
increased response rate (36% vs. 54%), a slight improvement in median
progression-free
survival (PFS) (4 vs. 6 months, p=0.02), but no improvement in median survival
(7.6 vs. 9.4
months, p=0.07). A second randomized trial evaluated the role of paclitaxel.
In this study,
patients are randomized to receive ifosfamide versus the combination of
ifosfamide plus
paclitaxel and showed an increased response rate (29% vs. 45%), improvement in
median
progression-free survival (3.6 vs. 5.8 months, p=0.03), and improvement in
median survival
(8.4 vs. 13.5 months, p=0.03). The use of ifosfamide is cumbersome and results
in
significant toxicity.
[0005] In a related disease, endometrial carcinoma, there have been several
randomized
studies addressing the issue of optimal therapy. These studies have focused on
three active
agents identified in phase II trials: doxorubicin, platinum agents, and
paclitaxel. In one
study, 281 women are randomized to doxorubicin alone (60 mg/m2) versus
doxorubicin (60
mg/m2) plus cisplatin (50 mg/m2) (AP). There is a statistically significant
advantage to
combination therapy with regard to response rate (RR) (25% versus 42%;
p=0.004) and PFS
(3.8 vs 5.7 months; HR 0.74 [95% CI 0.58, 0.94; p=0.14), although no
difference in OS is
observed (9 vs 9.2 months). Paclitaxel had significant single agent activity
with a response
rate of 36% in advanced or recurrent endometrial cancer. Thus 317 patients are
randomized
to paclitaxel and doxorubicin or the standard arm. This trial failed to
demonstrate a
significant difference in RR, PFS, or OS between the two arms, and AP remained
the
standard of care. However, since both platinum and paclitaxel had demonstrated
high single
agent activity, there is as strong interest in including paclitaxel and
cisplatin in a front-line
regimen for advanced and recurrent endometrial cancer. Subsequently, another
study
randomized 263 patients to AP versus TAP: doxorubicin (45 mg/m2) and cisplatin
(50
mg/m2) on day 1, followed by paclitaxel (160 mg/m2 IV over 3 hours) on day 2
(with G-CSF
support). TAP is superior to AP in terms of ORR (57% vs 34%; p<0.01), median
PFS (8.3 vs
5.3 months; p<0.01) and OS with a median of 15.3 (TAP) versus 12.3 months (AP)
(p=0.037). This improved efficacy came at the cost of increased toxicity.
[0006] Although there are limited therapeutic options for cancer treatment
generally,
recurrent, advanced or persistent uterine and ovarian cancers are especially
difficult to treat
because they can be refractory to standard chemotherapeutic treatments.
Therefore, there is a
need for effective cancer treatments generally, and for treatment of
recurrent, advanced, or
persistent cancers, such as ovarian or uterine cancers, in particular.
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BRIEF SUMMARY OF THE INVENTION
[00071 In one aspect, the present invention provides a method of treating
platinum-
sensitive recurrent ovarian cancer in a patient, comprising administering to
the patient having
ovarian cancer an effective amount of. (i) 4-iodo-3-nitrobenzamide or a
metabolite or a
pharmaceutically acceptable salt thereof; (ii) gemcitabine; and (iii)
carboplatin. The present
invention further provides a use of an effective amount of (i) 4-iodo-3-
nitrobenzamide or a
metabolite or a pharmaceutically acceptable salt thereof; (ii) gemcitabine;
and (iii)
carboplatin, for treating platinum-sensitive recurrent ovarian cancer in a
patient. The present
invention further provides a use of an effective amount of (i) 4-iodo-3-
nitrobenzamide or a
metabolite or a pharmaceutically acceptable salt thereof, (ii) gemcitabine;
and (iii)
carboplatin, for the preparation of (a) medicament(s) for treating platinum-
sensitive recurrent
ovarian cancer in a patient. The present invention further provides an
effective amount of (i)
4-iodo-3-nitrobenzamide or a metabolite or a pharmaceutically acceptable salt
thereof; (ii)
gemcitabine; and (iii) carboplatin, for use in treating platinum-sensitive
recurrent ovarian
cancer in a patient. In some embodiments, the effective amount is administered
over a 21-day
treatment cycle, wherein (i) the effective amount of carboplatin is
administered to the patient
at 4 mg/ml-minute (AUC 4) on day 1 of the treatment cycle; (ii) the effective
amount of
gemcitabine is administered to the patient at a dose of 1000 mg/m2 on days 1
and 8 of the
treatment cycle; and (iii) the effective amount of 4-iodo-3-nitrobenzamide or
a metabolite or
a pharmaceutically acceptable salt thereof is administered to the patient at a
dose of 5.6
mg/kg twice weekly on days 1, 4, 8, and 11 of the treatment cycle. In some
embodiments,
the effective amount produces at least one therapeutic effect selected from
the group
consisting of reduction in size of an ovarian tumor, reduction in metastasis,
complete
remission, partial remission, stable disease, increase in overall response
rate, or a pathologic
complete response. In some embodiments, a comparable clinical benefit rate
(CBR = CR
(complete remission) + PR (partial remission) + SD (stable disease) > 6
months) is obtained
compared to treatment with gemcitabine and carboplatin administered without 4-
iodo-3-
nitrobenzamide. In some embodiments, the improvement of clinical benefit rate
is about
20% or higher. In some embodiments, the therapeutic effect is an increase in
overall
response rate. In some embodiments, the overall response rate is greater than
40%. In some
embodiments, the overall response rate is greater than 50%. In some
embodiments, the
overall response rate is greater than 60%. In some embodiments, the method
further
comprises surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy,
viral
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therapy, RNA therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy,
immunotherapy, nanotherapy or a combination thereof. In some embodiments, the
method
further comprises administering to the patient gamma irradiation. In some
embodiments, the
platinum-sensitive recurrent ovarian cancer is selected from the group
consisting of epithelial,
germ cell, and stromal cell tumors. In some embodiments, the the platinum-
sensitive
recurrent ovarian cancer is metastatic.
[0008] In some embodiments, the platinum-sensitive recurrent ovarian cancer is
deficient
in homologous recombination DNA repair. In some embodiments, the homologous
recombination DNA repair-deficient platinum-sensitive recurrent ovarian cancer
is BRCA
deficient. In some embodiments, the BRCA-deficient platinum-sensitive
recurrent ovarian
cancer is BRCA I -deficient. In some embodiments, the BRCA-deficient platinum-
sensitive
recurrent ovarian cancer is BRCA2-deficient. In some embodiments, the BRCA-
deficient
platinum-sensitive recurrent ovarian cancer is both BRCAI-deficient and BRCA2-
deficient.
[0009] In one aspect, the present invention provides a method of treating
platinum-
sensitive ovarian cancer in a patient, comprising administering to the patient
having ovarian
cancer an effective amount of: (i) 4-iodo-3-nitrobenzamide or a metabolite or
a
pharmaceutically acceptable salt thereof; (ii) gemcitabine; and (iii)
carboplatin. In some
embodiments, the effective amount is administered over a 21-day treatment
cycle, wherein (i)
the effective amount of carboplatin is administered to the patient at 4 mg/ml-
minute (AUC 4)
on day 1 of the treatment cycle; (ii) the effective amount of gemcitabine is
administered to
the patient at a dose of 1000 mg/m2 on days 1 and 8 of the treatment cycle;
and (iii) the
effective amount of 4-iodo-3-nitrobenzamide or a metabolite or a
pharmaceutically
acceptable salt thereof is administered to the patient at a dose of 5.6 mg/kg
twice weekly on
days 1, 4, 8, and 11 of the treatment cycle. In some embodiments, the
effective amount
produces at least one therapeutic effect selected from the group consisting of
reduction in size
of an ovarian tumor, reduction in metastasis, complete remission, partial
remission, stable
disease, increase in overall response rate, or a pathologic complete response.
In some
embodiments, a comparable clinical benefit rate (CBR = CR (complete remission)
+ PR
(partial remission) + SD (stable disease) > 6 months) is obtained compared to
treatment with
gemcitabine and carboplatin administered without 4-iodo-3-nitrobenzamide. In
some
embodiments, the improvement of clinical benefit rate is about 20% or higher.
In some
embodiments, the therapeutic effect is an increase in overall response rate.
In some
embodiments, the overall response rate is greater than 40%. In some
embodiments, the
overall response rate is greater than 50%. In some embodiments, the overall
response rate is
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CA 02725026 2010-12-10
greater than 60%. In some embodiments, the method further comprises surgery,
radiation
therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,
DNA
therapy, adjuvant therapy, neoadjuvant therapy, immunotherapy, nanotherapy or
a
combination thereof. In some embodiments, the method further comprises
administering to
the patient gamma irradiation. In some embodiments, the platinum-sensitive
ovarian cancer
is selected from the group consisting of epithelial, germ cell, and stromal
cell tumors. In
some embodiments, the platinum-sensitive ovarian cancer is recurrent ovarian
cancer. In
some embodiments, the platinum-sensitive ovarian cancer is metastatic.
[0010] In some embodiments, the platinum-sensitive ovarian cancer is deficient
in
homologous recombination DNA repair. In some embodiments, the homologous
recombination DNA repair-deficient platinum-sensitive ovarian cancer is BRCA
deficient. In
some embodiments, the BRCA-deficient platinum-sensitive ovarian cancer is
BRCA1-
deficient. In some embodiments, the BRCA-deficient platinum-sensitive ovarian
cancer is
BRCA2-deficient. In some embodiments, the BRCA-deficient platinum-sensitive
ovarian
cancer is both BRCA1-deficient and BRCA2-deficient.
[0011] In another aspect is provided the use of the pharmaceutical
compositions
described herein for the manufacture of a medicament for treating platinum-
sensitive ovarian
cancer. For example, use as provided herein with respect to the methods
described herein.
[0012] In a further aspect is provided the use of 4-iodo-3-nitrobenzamide, a
metabolite
thereof, or a pharmaceutically acceptable salt or solvate thereof, in
combination with any one
of the antimetabolites and with any one of the platinum compounds described
herein, a
pharmaceutically acceptable salt or solvate thereof for the manufacture of a
medicament for
the treatment or prevention of platinum-sensitive ovarian cancer as described
herein.
[0013] In some aspects are provided synergistic compositions used for treating
platinum-
sensitive ovarian cancer in a patient comprising a) 4-iodo-3-nitrobenzamide,
or a metabolite
thereof, or a pharmaceutically acceptable salt or solvate thereof, b) an
antimetabolite, or
pharmaceutically acceptable salt or solvate thereof, and (c) a platinum
compound to said
patient, wherein the alkylating agent is selected from the group consisting of
citabme,
capecitabine, gemcitabine or valopicitabine), and wherein the platinum
compound is selected
from the group consisting of cisplatin; cis-diamminediaquoplatinum (11)-ion;
chloro(diethylenetriamine)-platinum (II) chloride; dichloro(ethylenediamine)-
platinum (II);
diammine(1,1-cyclobutanedicarboxylato) platinum (II) (carboplatin);
spiroplatin; iproplatin;
diammine(2-ethylmalonato)platinum (II); ethylenediaminemalonatoplatinum (II);
aqua(1,2-
diaminodicyclohexane)sulfatoplatinum (II); aqua(1,2-
CA 02725026 2010-12-10
diaminodicyclohexane)malonatoplatinum (II); (1,2-
diaminocyclohexane)malonatoplatinum
(II); (4-carboxyphthalato)(1,2-diaminocyclohexane) platinum (II); (1,2-
diaminocyclohexane)-
(isocitrato)platinum (II); (1,2-diaminocyclohexane)oxalatoplatinum (II);
ormiplatin;
tetraplatin; carboplatin, nedaplatin and oxaliplatin.
INCORPORATION BY REFERENCE
[0014] All publications and patent applications mentioned in this
specification are herein
incorporated by reference to the same extent as if each individual publication
or patent
application is specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE FIGURES
[0015] The novel features of the invention are set forth with particularity in
the appended
claims. A better understanding of the features and advantages of the present
invention will be
obtained by reference to the following detailed description that sets forth
illustrative
embodiments, in which the principles of the invention are utilized, and the
accompanying
drawings.
[0016] FIGURE 1 shows tumor response after 4 cycles of 4-iodo-3-nitrobenzamide
treatment in combination with topotecan in a patient with ovarian cancer.
[0017] FIGURE 2 shows PARP inhibition in peripheral mononuclear blood cells
(PMBCs) from patients receiving 4-iodo-3-nitrobenzamide.
DETAILED DESCRIPTION
[0018] As used herein, the term "platinum-sensitive" refers to a type of
ovarian cancer
(e.g., recurrent ovarian cancer). The current standard of care for first-line
chemotherapy of
ovarian cancer is a combination of a platinum compound (e.g., cisplatin,
carboplatin, and
oxaliplatin) with a taxane. The majority of newly-diagnosed ovarian cancer
patients will
respond to first-line platinum-based and paclitaxel chemotherapy. However, 50-
80% of the
patients who respond to this combination therapy will eventually relapse. See,
e.g., Herzog,
"Update on the role of topotecan in the treatment of recurrent ovarian
cancer," The
Oncologist 7(Suppl. 5):3-10 (2002). Patients who relapse within six months are
less likely to
respond to a second round of platinum-based therapy. Therefore, advanced
ovarian cancer
tumors that have recurred are classified as being "platinum-sensitive" if
relapse occurs more
than six months after the last dose of platinum-based therapy, "platinum-
resistant" if relapse
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CA 02725026 2010-12-10
occurs less than or equal to six months after the last dose of platinum-based
therapy, and
"platinum-refractory" if no response or disease regression occurs during
initial platinum-
based therapy.
[0019] As used herein "surgery" refers to any therapeutic or diagnostic
procedure that
involves methodical action of the hand or of the hand with an instrument, on
the body of a
human or other mammal, to produce a curative, remedial, or diagnostic effect.
[0020] "Radiation therapy" refers to exposing a patient to high-energy
radiation,
including without limitation x-rays, gamma rays, and neutrons. This type of
therapy includes
without limitation external-beam therapy, internal radiation therapy, implant
radiation,
brachytherapy, systemic radiation therapy, and radiotherapy.
[0021] "Chemotherapy" refers to the administration of one or more anti-cancer
drugs
such as, antineoplastic chemotherapeutic agents, chemopreventative agents,
and/or other
agents to a patient with platinum-sensitive ovarian cancer (e.g., recurrent
ovarian cancer) by
various methods, including intravenous, oral, intramuscular, intraperitoneal,
intravesical,
subcutaneous, transdermal, buccal, or inhalation or in the form of a
suppository. Unless
clearly dictated otherwise by context, "chemotherapy" as used herein is not
intended to refer
to the administration of 4-iodo-3-nitrobenzamide, an antimetabolite (e.g.,
gemcitabine), and a
platinum compound (e.g., carboplatin) as described herein. Chemotherapy may be
given prior
to surgery to shrink a large tumor prior to a surgical procedure to remove it,
prior to radiation
therapy, or after surgery and/or radiation therapy to prevent the growth of
any remaining
ovarian cancer cells in the body. Chemotherapy may also occur during the
course of
radiation therapy.
[0022] The terms "effective amount" or "pharmaceutically effective amount"
refer to a
sufficient amount of an agent to provide the desired biological, therapeutic,
and/or
prophylactic result. That result can be reduction and/or alleviation of one or
more of the
signs, symptoms, or causes of a disease, or any other desired alteration of a
biological system.
For example, an "effective amount" for therapeutic uses is the amount of a) 4-
iodo-3-
nitrobenzamide or a metabolite thereof or a pharmaceutically acceptable salt
or solvate
thereof; b) an antimetabolite (e.g., gemcitabine), or pharmaceutically
acceptable salt or
solvate thereof; and c) a platinum compound (e.g., carboplatin) provided
herein, or a
composition comprising a) 4-iodo-3-nitrobenzamide or a metabolite thereof or a
pharmaceutically acceptable salt or solvate thereof; b) an antimetabolite
(e.g., gemcitabine),
or pharmaceutically acceptable salt or solvate thereof; and c) a platinum
compound (e.g.,
carboplatin) provided herein required to provide a clinically significant
decrease in the
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CA 02725026 2010-12-10
platinum-sensitive ovarian cancer (e.g., recurrent ovarian cancer) or slowing
of progression
of the platinum-sensitive ovarian cancer (e.g., recurrent ovarian cancer).
[0023] "Metabolite" refers to a compound produced through any in vitro or in
vivo
metabolic process which results in a product that is different in structure
than that of the
starting compound. In other words, the term "metabolite" includes the
metabolite compounds
of 4-iodo-3-nitrobenzamide. A metabolite can include a varying number or types
of
substituents that are present at any position relative to a precursor
compound. In addition, the
terms "metabolite" and "metabolite compound" are used interchangeably herein.
[0024] By "pharmaceutically acceptable" is meant a material which is not
biologically or
otherwise undesirable, i.e., the material may be administered to a patient
without causing any
undesirable biological effects or interacting in a deleterious manner with any
of the
components of the composition in which it is contained.
[0025] The term "treating" and its grammatical equivalents as used herein
include
achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic
benefit is meant
eradication or amelioration of the underlying disorder being treated. For
example, in a
patient having platinum-sensitive ovarian cancer (e.g., recurrent ovarian
cancer), therapeutic
benefit includes eradication or amelioration of the underlying ovarian cancer,
e.g., slowing of
progression of the ovarian cancer. Also, a therapeutic benefit is achieved
with the eradication
or amelioration of one or more of the physiological symptoms associated with
the underlying
disorder (e.g., ovarian cancer) such that an improvement is observed in the
patient,
notwithstanding the fact that the patient may still be afflicted with the
underlying disorder
(e.g., ovarian cancer). For prophylactic benefit, a method of the invention
may be performed
on, or a composition of the invention administered to a patient at risk of
developing platinum-
sensitive ovarian cancer (e.g., recurrent ovarian cancer), or to a patient
reporting one or more
of the physiological symptoms of platinum-sensitive ovarian cancer (e.g.,
recurrent ovarian
cancer), even though a diagnosis of platinum-sensitive ovarian cancer (e.g.,
recurrent ovarian
cancer) may not have been made. In some embodiments, the patient being treated
has been
diagnosed with a platinum-sensitive ovarian cancer (e.g., recurrent ovarian
cancer) described
herein.
[0026] Reference to "about" a value or parameter herein includes (and
describes)
variations that are directed to that value or parameter per se. For example,
description
referring to "about X" includes description of "X".
[0027] As used herein and in the appended claims, the singular forms "a,"
"or," and "the"
include plural referents unless the context clearly dictates otherwise. It is
understood that
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CA 02725026 2010-12-10
aspects and variations of the invention described herein include "consisting"
and/or
"consisting essentially of" aspects and variations.
Treatment of Ovarian Cancer
[0028] Ovarian cancer is the leading cause of death from gynecologic
malignancy. It is
the eighth most common cancer in women, with approximately 21,550 new
diagnoses and
14,600 deaths in the United States in 2009. Ovarian cancer is difficult to
detect in its early
stages; only about 20 percent of ovarian cancers are found before tumor growth
has spread
into adjacent tissues. By the time ovarian cancer has progressed to later
stages of the disease,
the long-term prognosis is generally poor, with a five-year survival rate of -
30%. Despite
high initial response to the first line neoadjuvant chemotherapy-a combination
of a platinum
compound and a taxane-recurrence is common, with a median progression-free
survival of
about eighteen months.
[0029] There are three basic types of ovarian tumors: epithelial, germ cell,
and stromal
cell tumors. Epithelial tumors start from the cells that cover the outer
surface of the ovary;
most ovarian tumors are epithelial cell tumors. Germ cell tumors start from
the cells that
produce the eggs. Stromal tumors start from cells that hold the ovary together
and make the
female hormones.
[0030] A significant risk factor for ovarian cancer includes deficiencies in
DNA repair
via homologous recombination, such as mutations in the BRCA1 or BRCA2 gene.
Those
genes were originally identified in families with multiple cases of breast
cancer, but have
been associated with approximately 5 to 10 percent of ovarian cancers.
[0031] Possible treatments for ovarian cancer include surgery, immunotherapy,
chemotherapy, hormone therapy, radiation therapy, or a combination thereof.
Surgical
procedures for the treatment of ovarian cancer include debulking, and a
unilateral or bilateral
oophorectomy and/or a unilateral or bilateral salpigectomy. Anti-cancer drugs
that have also
been used to treat ovarian cancer include cyclophosphamide, etoposide,
altretamine, and
ifosfamide. Hormone therapy with the drug tamoxifen is also used to shrink
ovarian tumors.
Radiation therapy optionally includes external beam radiation therapy and/or
brachytherapy.
[0032] In one aspect, provided herein are methods of treating platinum-
sensitive recurrent
ovarian cancer in a patient, comprising administering to the patient a PARP
inhibitor, an
antimetabolite, and a platinum compound. In some embodiments, the PARP
inhibitor is a
PARP-1 inhibitor. In some embodiments, the PARP-1 inhibitor is 4-iodo-3-
nitrobenzamide
or a metabolite or a pharmaceutically acceptable salt thereof. In some
embodiments, the
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CA 02725026 2010-12-10
antimetabolite is selected from the group consisting of citabine,
capecitabine, gemcitabine or
valopicitabine. In some embodiments, the antimetabolite is gemcitabine. In
some
embodiments, the platinum compound is selected from the group consisting of
cisplatin; cis-
diamminediaquoplatinum (II)-ion; chloro(diethylenetriamine)-platinum (II)
chloride;
dichloro(ethylenediamine)-platinum (II); diammine(1,1-
cyclobutanedicarboxylato) platinum
(II) (carboplatin); spiroplatin; iproplatin; diammine(2-ethylmalonato)platinum
(II);
ethylenediaminemalonatoplatinum (II); aqua(1,2-
diaminodicyclohexane)sulfatoplatinum (II);
aqua(1,2-diaminodicyclohexane)malonatoplatinum (II); (1,2-
diaminocyclohexane)malonatoplatinum (II); (4-carboxyphthalato)(1,2-
diaminocyclohexane)
platinum (II); (1,2-diaminocyclohexane)-(isocitrato)platinum (II); (1,2-
diaminocyclohexane)oxalatoplatinum (II); ormaplatin; tetraplatin; carboplatin,
nedaplatin and
oxaliplatin, and preferred is carboplatin or oxaliplatin. In some embodiments,
the platinum
compound is carboplatin.
[0033] In some embodiments, the method further comprises surgery, radiation
therapy,
chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, DNA
therapy,
adjuvant therapy, neoadjuvant therapy, immunotherapy, nanotherapy or a
combination
thereof. In some embodiments, the method further comprises administering to
the patient
gamma irradiation. In some embodiments, the platinum-sensitive recurrent
ovarian cancer is
selected from the group consisting of epithelial, germ cell, and stromal cell
tumors. In some
embodiments, the the platinum-sensitive recurrent ovarian cancer is
metastatic.
[0034] In some embodiments, the platinum-sensitive recurrent ovarian cancer is
deficient
in homologous recombination DNA repair. In some embodiments, the homologous
recombination DNA repair-deficient platinum-sensitive recurrent ovarian cancer
is BRCA
deficient. In some embodiments, the BRCA-deficient platinum-sensitive
recurrent ovarian
cancer is BRCA1-deficient. In some embodiments, the BRCA-deficient platinum-
sensitive
recurrent ovarian cancer is BRCA2-deficient. In some embodiments, the BRCA-
deficient
platinum-sensitive recurrent ovarian cancer is both BRCAI-deficient and BRCA2-
deficient.
[0035] In some embodiments, at least one therapeutic effect is obtained, said
at least one
therapeutic effect being reduction in size of an ovarian tumor, reduction in
metastasis,
complete remission, partial remission, pathologic complete response, increase
in overall
response rate or stable disease. In some embodiments, an improvement of
clinical benefit
rate (CBR = CR + PR + SD > 6 months) is obtained as compared to treatment
without the
PARP inhibitor. In some embodiments, the improvement of clinical benefit rate
is at least
about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more. In some embodiments, the
CA 02725026 2010-12-10
therapeutic effect is an increase in overall response rate. In some
embodiments, the increase
in overall response rate is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.
[0036] In some embodiments, the platinum-sensitive ovarian cancer is a
metastatic
ovarian cancer. In some embodiments, a deficiency in a BRCA gene is detected
in the
ovarian cancer patient. In some embodiments, the BRCA gene is BRCA 1. In other
embodiments, the BRCA gene is BRCA-2. In yet other embodiments, the BRCA gene
is
BRCA-1 and BRCA-2. In other embodiments, the deficiency is a genetic defect in
the
BRCA gene. In some embodiments, the genetic defect is a mutation, insertion,
substitution,
duplication or deletion of the BRCA gene.
[0037] In some embodiments, the methods for treating platinum-sensitive
recurrent
ovarian cancer further comprise administering a PARP inhibitor in combination
with an anti-
tumor agent. In some embodiments, the anti-tumor agent is an antitumor
alkylating agent,
antitumor antimetabolite, antitumor antibiotics, plant-derived antitumor
agent, antitumor
platinum complex, antitumor camptothecin derivative, antitumor tyrosine kinase
inhibitor,
monoclonal antibody, interferon, biological response modifier, hormonal anti-
tumor agent,
anti-tumor viral agent, angiogenesis inhibitor, differentiating agent, or
other agent that
exhibits anti-tumor activities, or a pharmaceutically acceptable salt thereof.
In some
embodiments, the platinum complex is cisplatin, carboplatin, oxaplatin or
oxaliplatin. In
some embodiments, the antimetabolite is citabine, capecitabine, gemcitabine or
valopicitabine. In some embodiments, the methods further comprise
administering to the
patient a PARP inhibitor in combination with more than one anti-tumor agent.
In some
embodiments, the anti-tumor agent is administered prior to, concomitant with
or subsequent
to administering the PARP inhibitor. In some embodiments, the anti-tumor agent
is an anti-
angiogenic agent, such as Avastin or a receptor tyrosine kinase inhibitor
including but not
limited to Sutent, Nexavar, Recentin, ABT-869, and Axitinib. In some
embodiments, the anti-
tumor agent is a topoisomerase inhibitor including but not limited to
irinotecan, topotecan, or
camptothecin. In some embodiments, the anti-tumor agent is a taxane including
but not
limited to paclitaxel, docetaxel and Abraxane. In some embodiments, the anti-
tumor agent is
an agent targeting Her-2, Herceptin or lapatinib. In some embodiments, the
anti-tumor agent
is a hormone analog, for example, progesterone. In some embodiments, the anti-
tumor agent
is tamoxifen, a steroidal aromatase inhibitor, a non-steroidal aromatase
inhibitor, or
Fulvestrant. In some embodiments, the anti-tumor agent is an agent targeting a
growth factor
receptor. In some embodiments, such agent is an inhibitor of epidermal growth
factor
receptor (EGFR) including but not limited to Cetuximab and Panitumimab. In
some
11
CA 02725026 2010-12-10
embodiments, the agent targeting a growth factor receptor is an inhibitor of
insulin-like
growth factor 1 (IGF-1) receptor (IGFIR) such as CP-751871. In other
embodiments, the
method further comprises surgery, radiation therapy, chemotherapy, gene
therapy, DNA
therapy, adjuvant therapy, neoadjuvant therapy, viral therapy, RNA therapy,
immunotherapy,
nanotherapy or a combination thereof.
[0038] In some embodiments, the methods for treating platinum-sensitive
recurrent
ovarian cancer further comprise administering a PARP inhibitor in combination
with an anti-
tumor agent. In some embodiments, the anti-tumor agent is an antitumor
alkylating agent,
antitumor antimetabolite, antitumor antibiotics, anti-tumor viral agent, plant-
derived
antitumor agent, antitumor platinum complex, antitumor camptothecin
derivative, antitumor
tyrosine kinase inhibitor, monoclonal antibody, interferon, biological
response modifier,
hormonal anti-tumor agent, angiogenesis inhibitor, differentiating agent, or
other agent that
exhibits anti-tumor activities, or a pharmaceutically acceptable salt thereof.
In some
embodiments, the platinum complex is cisplatin, carboplatin, oxaplatin or
oxaliplatin. In
some embodiments, the antimetabolite is citabine, capecitabine, gemcitabine or
valopicitabine. In some embodiments, the methods further comprise
administering to the
patient a PARP inhibitor in combination with more than one anti-tumor agent.
In some
embodiments, the anti-tumor agent is administered prior to, concomitant with
or subsequent
to administering the PARP inhibitor. In other embodiments, the method further
comprises
surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, adjuvant
therapy,
neoadjuvant therapy, viral therapy, RNA therapy, immunotherapy, nanotherapy or
a
combination thereof.
[0039] In some embodiments, the treatment comprises a treatment cycle of at
least 11
days, i.e. about 11 to about 30 days in length, wherein on from 1 to 10
separate days of the
cycle, the patient receives about 1 to about 100 mg/kg of 4-iodo-3-
nitrobenzamide or a molar
equivalent of a metabolite thereof. In some embodiments, on from 1 to 10
separate days of
the cycle, the patient receives about 1 to about 50 mg/kg of 4-iodo-3-
nitrobenzamide or a
molar equivalent of a metabolite thereof. In some embodiments, on from 1 to 10
separate
days of the cycle, the patient receives about 1, 2, 3, 4, 6, 8 or 10, 12, 14,
16, 18 or 20 mg/kg
of 4-iodo-3-nitrobenzamide.
[0040] Some embodiments described herein provide a method of treating ovarian
cancer
in a patient having a deficiency in a BRCA gene, comprising during a 21 day
treatment cycle
on days 1, 4, 8 and 11 of the cycle, administering to the patient about 10 to
about 100 mg/kg
of 4-iodo-3-nitrobenzamide or a molar equivalent of a metabolite thereof. In
some
12
CA 02725026 2010-12-10
embodiments, the 4-iodo-3-nitrobenzamide is administered orally or as a
parenteral injection
or infusion, or inhalation.
[0041] Some embodiments described herein provide a method of treating ovarian
cancer
in a patient having a deficiency in a BRCA gene, comprising: (a) establishing
a treatment
cycle of about 10 to about 30 days in length; (b) on from 1 to 10 separate
days of the cycle,
administering to the patient about 1 mg/kg to about 50 mg/kg of 4-iodo-3-
nitrobenzamide, or
a molar equivalent of a metabolite thereof. In some embodiments, the 4-iodo-3-
nitrobenzamide is administered orally or as a parenteral injection or
infusion, or inhalation.
[0042] Some embodiments provided herein include a method of treating ovarian
cancer
in a patient in need of such treatment, comprising: (a) obtaining a sample
from the patient;
(b) testing the sample to determine if there is a deficiency in a BRCA gene;
(c) if the testing
indicates that the patient has a deficiency in a BRCA gene, treating the
patient with at least
one PARP inhibitor; and (d) if the testing does not indicate that the patient
has a deficiency in
a BRCA gene, selecting a different treatment option. In some embodiments, at
least one
therapeutic effect is obtained, said at least one therapeutic effect being
reduction in size of an
ovarian tumor, reduction in metastasis, complete remission, partial remission,
pathologic
complete response, increase in overall response rate, or stable disease. In
some
embodiments, an improvement of clinical benefit rate (CBR = CR + PR + SD > 6
months) is
obtained as compared to treatment without the PARP inhibitor. In some
embodiments, the
clinical benefit rate is at least about 30%. In some embodiments, the PARP
inhibitor is a
PARP- 1 inhibitor. In other embodiments, the PARP-1 inhibitor is a benzamide
or a
metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-
nitrobenzamide or a
metabolite thereof. In some embodiments, the sample is a tissue or bodily
fluid sample. In
some embodiments, the sample is a tumor sample, a blood sample, a blood plasma
sample, a
peritoneal fluid sample, an exudate or an effusion. In some embodiments, the
ovarian cancer
is a metastatic ovarian cancer. In some embodiments, the BRCA gene is BRCA-1.
In other
embodiments, the BRCA gene is BRCA-2. In some embodiments, the BRCA gene is
BRCA-
1 and BRCA-2. In other embodiments, the deficiency is a genetic defect in the
BRCA gene.
In some embodiments, the genetic defect is a mutation, insertion,
substitution, duplication or
deletion of the BRCA gene.
[0043] Some embodiments provide a method of treating ovarian cancer in a
patient,
comprising: (a) testing a sample from the patient for PARP expression; and (b)
if the PARP
expression exceeds a predetermined level, administering to the patient at
least one PARP
inhibitor. In some embodiments, at least one therapeutic effect is obtained,
said at least one
13
CA 02725026 2010-12-10
therapeutic effect being reduction in size of an ovarian tumor, reduction in
metastasis,
complete remission, partial remission, pathologic complete response, or stable
disease. In
some embodiments, an improvement of clinical benefit rate (CBR = CR + PR + SD
> 6
months) is obtained as compared to treatment without the PARP inhibitor. In
some
embodiments, the improvement of clinical benefit rate is at least about 30%.
In some
embodiments, the PARP inhibitor is a PARP-1 inhibitor. In other embodiments,
the PARP-1
inhibitor is a benzamide or a metabolite thereof. In some embodiments, the
benzamide is 4-
iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the
ovarian cancer is a
metastatic ovarian cancer.
Treatment of Uterine and Endometrial Cancer
[0044] Malignant uterine neoplasms containing both carcinomatous and
sarcomatous
elements are designated in the World Health Organization (WHO) classification
of uterine
neoplasms as carcinosarcomas. An alternative designation is malignant mixed
Mullerian
tumor (MMMT). Most uterine carcinosarcomas are monoclonal, with the
carcinomatous
element being the key element and the sarcomatous component derived from the
carcinoma
or from a stem cell that undergoes divergent differentiation (i.e.,
metaplastic carcinomas).
The sarcomatous component is either homologous (composed of tissues normally
found in
the uterus) or heterologous (containing tissues not normally found in the
uterus, most
commonly malignant cartilage or skeletal muscle).
[0045] Previous studies investigating a number of single agents in
carcinosarcoma of the
uterus have reported the following response rates: etoposide (6.5%);
doxorubicin (9.8%);
cisplatin (18%); ifosfamide (32.2%); paclitaxel (18.2%); and topotecan (10%).
Thus the
three most active agents discovered to date include cisplatin, ifosfamide, and
paclitaxel. A
randomized phase III trial comparing ifosfamide to ifosfamide plus cisplatin
showed an
increased response rate (36% vs. 54%), a slight improvement in median
progression-free
survival (4 vs. 6 months, p=0.02), but no improvement in median survival (7.6
vs. 9.4
months, p=0.07). A second randomized trial evaluated the role of paclitaxel.
In this study,
patients are randomized to receive ifosfamide versus the combination of
ifosfamide plus
paclitaxel and showed an increased response rate (29% vs. 45%), improvement in
median
progression-free survival (3.6 vs. 5.8 months, p=0.03), and improvement in
median survival
(8.4 vs. 13.5 months, p=0.03). The use of ifosfamide is cumbersome and results
in
significant toxicity.
14
CA 02725026 2010-12-10
[0046] In a highly related disease, endometrial carcinoma, there have been
several
randomized studies addressing the issue of optimal therapy. These studies have
focused on
three active agents identified in phase II trials: doxorubicin, platinum
agents, and paclitaxel.
In one study, 281 women are randomized to doxorubicin alone (60 mg/m2) versus
doxorubicin (60 mg/m2) plus cisplatin (50 mg/m2) (AP). There is a
statistically significant
advantage to combination therapy with regard to response rate (RR) (25% versus
42%;
p=0.004) and PFS (3.8 vs 5.7 months; HR 0.74 [95% Cl 0.58, 0.94; p=0.14),
although no
difference in OS is observed (9 vs 9.2 months). Paclitaxel had significant
single agent
activity with a response rate of 36% in advanced or recurrent endometrial
cancer. Thus 317
patients are randomized to paclitaxel and doxorubicin or the standard arm.
This trial failed to
demonstrate a significant difference in RR, PFS, or OS between the two arms,
and AP
remained the standard of care. However, since both platinum and paclitaxel had
demonstrated high single agent activity, there is as strong interest in
including paclitaxel and
cisplatin in a front-line regimen for advanced and recurrent endometrial
cancer.
Subsequently, another study randomized 263 patients to AP versus TAP:
doxorubicin (45
mg/m2) and cisplatin (50 mg/m2) on day 1, followed by paclitaxel (160 mg/m2 IV
over 3
hours) on day 2 (with G-CSF support). TAP is superior to AP in terms of ORR
(57% vs
34%; p<0.01), median PFS (8.3 vs 5.3 months; p<0.01) and OS with a median of
15.3 (TAP)
versus 12.3 months (AP) (p=0.037). This improved efficacy, however, came at
the cost of
increased toxicity.
Uterine Tumors
[0047] Uterine tumors consist of the group of neoplasm that can be localized
at the
corpus, isthmus (the transition between the endocervix and uterine corpus) and
cervix. The
fallopian tubes and uterine ligaments may also undergo tumor transformation.
Uterine tumors
may affect the endometrium, muscles or other supporting tissue. Uterine tumors
are
histologically and biologically different and can be divided into several
types. Uterine tumors
may be histologically typed according to several classification systems. Those
used most
frequently are based on the WHO (World Health Organization) International
Histological
Classification of Tumours and on the ISGYP (International Society of
Gynecological
Pathologists). The most widely-accepted staging system is the FIGO
(International
Federation of Gynecology and Obstetrics) one.
CA 02725026 2010-12-10
Classification
[0048] According to the World Health Organization ("WHO"), the main categories
of
uterine and cervical cancers are: epithelial tumors; mesenchymal tumors; mixed
epithelial
and mesenchymal tumors; and secondary tumors. The main uterine corpus
categories, once
again according to the WHO, are: epithelial tumors, mesenchymal tumors, mixed
epithelial
and mesenchymal tumors, trophoblastic tumors, and secondary tumors. Uterine
cancer is the
most common, specifically endometrial cancer of the uterine corpus.
[0049] Uterine Corpus Neoplasia. The most common uterine corpus malignancy is
the
endometrial carcinoma (approximately 95%); sarcomas represent only 4% and
heterologous
tumors such as rhabdomyosarcomas, osteosarcomas and chondrosarcomas the
remaining 1%.
[0050] Endometrial carcinoma has several subtypes that based on origin,
differentiation,
genetic background and clinical outcome. Endometrial carcinoma is defined as
an epithelial
tumor, usually with glandular differentiation, arising in the endometrium and
which has the
potential to invade the myometrium and spread to distant sites. Endometrial
carcinoma can be
classified as endometrioid adenocarcinoma, serous carcinoma, clear cell
carcinoma,
mucinous carcinoma, serous carcinoma, mixed types of carcinoma, and
undifferentiated
carcinoma. Endometrial carcinoma is an heterogeneous entity, comprising of:
type I:
endometrioid carcinoma : pre- and perimenopausal, estrogen dependent,
associated to
endometrial hyperplasia, low grade, indolent behaviour, representing about 80
% of the cases;
type II: serous carcinoma : post-menopausal, estrogen independent, associated
to atrophic
endometrium, high grade, aggressive behaviour, representing about 10 % of the
cases.
Among other histologic types, type I includes mucinous and secretory
carcinomas, whereas
type II includes clear-cell carcinomas and adenosquamous carcinomas (Gurpide
E, J Natl
Cancer Inst 1991; 83: 405-416; Blaustein's Pathology of the Female Genital
Tract, Kurman
R.J. 4th ed. Springer-Verlag. New-York 1994).
[0051] Uterine Cervix Neoplasia. Worldwide, invasive cervical cancer is the
second
most common female malignancy after breast cancer, with 500,000 new cases
diagnosed each
year. Uterine cervix cancers has several subtypes such as epithelial neoplasia
and
mesenchymal neoplasia.
Etiology
[0052] Carcinomas of the uterine cervix are thought to arise from precursor
lesions, and
different subtypes of human papilloma virus (HPV) are major etiological
factors in disease
pathogenesis.
16
CA 02725026 2010-12-10
[0053] Heterogeneity of uterine tumors provide a challenge to find and
optimize the
therapy to treat and cure these types of cancers and chemotherapeutic agent
that are
efficacious for other cancers are not efficacious for uterine tumors such as
endometrial
cancer. One of the examples could be Tamoxifen. Tamoxifen, a selective
estrogen receptor
(ER) modulator, is the most widely prescribed hormonal therapy treatment for
breast cancer.
Despite the benefits of tamoxifen therapy, almost all tamoxifen-responsive
breast cancer
patients develop resistance to therapy. In addition, tamoxifen displays
estrogen-like effects in
the endometrium increasing the incidence of endometrial cancer (Fisher B,
Costantino JP,
Redmond CK, et al., J Natl Cancer Inst 1994; 86:527-37; Shah YM, et al., Mol
Cancer Ther.
2005 Aug;4(8):1239-49).
[0054] In patients with persistent or recurrent nonsquamous cell carcinoma of
the cervix,
the study was undertaken by Gynecologic Oncology Group to estimate the
antitumor activity
of tamoxifen (L. R. Bigler, J. et al., (2004) International Journal of
Gynecological Cancer 14
(5), 871-874). Tamoxifen citrate is administered at a dose of 10 mg per orally
twice a day
until disease progression or unacceptable side effects prevented further
therapy. A total of 34
patients (median age: 49 years) are registered to this trial; two are declared
ineligible. Thirty-
two patients are evaluable for adverse effects and 27 are evaluable for
response. There are
only six grades 3 and 4 adverse effects reported: leukopenia (in one patient),
anemia (in two),
emesis (in one), gastrointestinal distress (in one), and neuropathy (in one).
The objective
response rate is 11.1%, with one complete and two partial responses. In
conclusion,
tamoxifen appears to have minimal activity in nonsquamous cell carcinoma of
the cervix.
[0055] Accordingly, some embodiments described herein provide a method of
treating
uterine cancer or endometrial cancer in a patient, comprising administering to
the patient at
least one PARP inhibitor. In some embodiments, at least one therapeutic effect
is obtained,
said at least one therapeutic effect being reduction in size of a uterine
tumor, reduction in
metastasis, complete remission, partial remission, pathologic complete
response, or stable
disease. In some embodiments, an improvement of clinical benefit rate (CBR =
CR + PR +
SD > 6 months) is obtained as compared to treatment without the PARP
inhibitor. In some
embodiments, the improvement of clinical benefit rate is at least about 30%.
In some
embodiments, the PARP inhibitor is a PARP-1 inhibitor. In other embodiments,
the PARP-1
inhibitor is a benzamide or a metabolite thereof. In some embodiments, the
benzamide is 4-
iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the
uterine cancer is a
metastatic uterine cancer. In some embodiments, the uterine cancer is
recurrent, advanced or
persistent.
17
CA 02725026 2010-12-10
[0056] In some embodiments, the methods for treating uterine cancer or
endometrial
cancer further comprise administering a PARP inhibitor in combination with an
anti-tumor
agent. In some embodiments, the anti-tumor agent is an antitumor alkylating
agent,
antitumor antimetabolite, antitumor antibiotics, plant-derived antitumor
agent, antitumor
platinum complex, antitumor camptothecin derivative, antitumor tyrosine kinase
inhibitor,
monoclonal antibody, interferon, biological response modifier, hormonal anti-
tumor agent,
anti-tumor viral agent, angiogenesis inhibitor, differentiating agent, or
other agent that
exhibits anti-tumor activities, or a pharmaceutically acceptable salt thereof.
In some
embodiments, the platinum complex is cisplatin, carboplatin, oxaplatin or
oxaliplatin. In
some embodiments, the antimetabolite is citabine, capecitabine, gemcitabine or
valopicitabine. In some embodiments, the methods further comprise
administering to the
patient a PARP inhibitor in combination with more than one anti-tumor agent.
In some
embodiments, the anti-tumor agent is administered prior to, concomitant with
or subsequent
to administering the PARP inhibitor. In some embodiments, the anti-tumor agent
is an anti-
angiogenic agent, such as Avastin or a receptor tyrosine kinase inhibitor
including but not
limited to Sutent, Nexavar, Recentin, ABT-869, and Axitinib. In some
embodiments, the anti-
tumor agent is a topoisomerase inhibitor including but not limited to
irinotecan, topotecan, or
camptothecin. In some embodiments, the anti-tumor agent is a taxane including
but not
limited to paclitaxel, docetaxel and Abraxane. In some embodiments, the anti-
tumor agent is
an agent targeting Her-2, e.g., Herceptin or lapatinib. In some embodiments,
the anti-tumor
agent is a hormone analog, for example, progesterone. In some embodiments, the
anti-tumor
agent is tamoxifen, a steroidal aromatase inhibitor, a non-steroidal aromatase
inhibitor, or
Fulvestrant. In some embodiments, the anti-tumor agent is an agent targeting a
growth factor
receptor. In some embodiments, such agent is an inhibitor of epidermal growth
factor
receptor (EGFR) including but not limited to Cetuximab and Panitumimab. In
some
embodiments, the agent targeting a growth factor receptor is an inhibitor of
insulin-like
growth factor 1 (IGF-1) receptor (IGF1R) such as CP-751871. In other
embodiments, the
method further comprises surgery, radiation therapy, chemotherapy, gene
therapy, DNA
therapy, adjuvant therapy, neoadjuvant therapy, viral therapy, RNA therapy,
immunotherapy,
nanotherapy or a combination thereof.
[0057] In some embodiments, the treatment comprises a treatment cycle of at
least 11
days, i.e. about 11 to about 30 days in length, wherein on from 1 to 10
separate days of the
cycle, the patient receives about 1 to about 100 mg/kg of 4-iodo-3-
nitrobenzamide or a molar
equivalent of a metabolite thereof. In some embodiments, on from 1 to 10
separate days of
18
CA 02725026 2010-12-10
the cycle, the patient receives about 1 to about 50 mg/kg of 4-iodo-3-
nitrobenzamide or a
molar equivalent of a metabolite thereof. In some embodiments, on from 1 to 10
separate
days of the cycle, the patient receives about 1, 2, 3, 4, 6, 8 or 10, 12, 14,
16, 18 or 20 mg/kg
of 4-iodo-3-nitrobenzamide.
[0058] Some embodiment described herein provide a method of treating uterine
cancer or
endometrial cancer in a patient, comprising during a 21 day treatment cycle on
days 1, 4, 8
and 11 of the cycle, administering to the patient about 1 to about 100 mg/kg
of 4-iodo-3-
nitrobenzamide or a molar equivalent of a metabolite thereof. In some
embodiments, the 4-
iodo-3-nitrobenzamide is administered orally or as a parenteral injection or
infusion, or
inhalation.
[0059] Some embodiments described herein provide a method of treating uterine
cancer
or endometrial cancer in a patient, comprising: (a) establishing a treatment
cycle of about 10
to about 30 days in length; (b) on from 1 to 10 separate days of the cycle,
administering to the
patient about 1 mg/kg to about 100 mg/kg of 4-iodo-3-nitrobenzamide, or a
molar equivalent
of a metabolite thereof. In some embodiments, the 4-iodo-3-nitrobenzamide is
administered
orally or as a parenteral injection or infusion, or inhalation.
[0060] Some embodiments provided herein include a method of treating uterine
cancer in
a patient in need of such treatment, comprising: (a) obtaining a sample from
the patient; (b)
determining if the uterine cancer is recurrent, persistent or advanced; (c) if
the testing
indicates that the uterine cancer is recurrent, persistent or advanced,
treating the patient with
at least one PARP inhibitor; and (d) if the testing does not indicate that the
patient has a
uterine cancer that is recurrent, persistent or advanced, selecting a
different treatment option.
In some embodiments, at least one therapeutic effect is obtained, said at
least one therapeutic
effect being reduction in size of a uterine tumor, reduction in metastasis,
complete remission,
partial remission, pathologic complete response, or stable disease. In some
embodiments, an
improvement of clinical benefit rate (CBR = CR + PR + SD > 6 months) is
obtained as
compared to treatment without the PARP inhibitor. In some embodiments, the
clinical
benefit rate is at least about 30%. In some embodiments, the PARP inhibitor is
a PARP-1
inhibitor. In other embodiments, the PARP-1 inhibitor is a benzamide or a
metabolite
thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a
metabolite
thereof. In some embodiments, the sample is a tissue or bodily fluid sample.
In some
embodiments, the sample is a tumor sample, a blood sample, a blood plasma
sample, a
peritoneal fluid sample, an exudate or an effusion. In some embodiments, the
uterine cancer
is a metastatic uterine cancer.
19
CA 02725026 2010-12-10
[0061] Some embodiments provide a method of treating uterine cancer,
endometrial
cancer, or ovarian cancer in a patient, comprising: (a) testing a sample from
the patient for
PARP expression; and (b) if the PARP expression exceeds a predetermined level,
administering to the patient at least one PARP inhibitor. In some embodiments,
at least one
therapeutic effect is obtained, said at least one therapeutic effect being
reduction in size of a
uterine tumor, reduction in metastasis, complete remission, partial remission,
pathologic
complete response, or stable disease. In some embodiments, an improvement of
clinical
benefit rate (CBR = CR + PR + SD > 6 months) is obtained as compared to
treatment without
the PARP inhibitor. In some embodiments, the improvement of clinical benefit
rate is at least
about 30%. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In
other
embodiments, the PARP-1 inhibitor is a benzamide or a metabolite thereof. In
some
embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof.
In some
embodiments, the uterine cancer is a metastatic uterine cancer. In some
embodiments, the
ovarian cancer is a metastatic ovarian cancer.
[0062] Thus, embodiments provided herein comprise treating a patient with at
least one
of which is a PARP inhibitor, wherein the PARP inhibitor is optionally a PARP-
1 inhibitor.
In some embodiments, one or more of these substances may be capable of being
present in a
variety of physical forms-e.g., free base, salts (especially pharmaceutically
acceptable
salts), hydrates, polymorphs, solvates, etc. Unless otherwise qualified
herein, use of a
chemical name is intended to encompass all physical forms of the named
chemical. For
example, recitation of 4-iodo-3-nitrobenzamide, without further qualification,
is intended to
generically encompass the free base as well as all pharmaceutically acceptable
salts,
polymorphs, hydrates, etc. Where it is intended to limit the disclosure or
claims to a
particular physical form of a compound, this will be clear from the context of
the passage or
claim in which the reference to the compound appears.
[0063] In some embodiments, the disclosure herein provides a method of
treating uterine
cancer, endometrial cancer, or ovarian cancer in a patient, comprising
administering to the
patient a combination of at least one anti-tumor agent and at least one PARP
inhibitor. In
some embodiments, at least one therapeutic effect is obtained, said at least
one therapeutic
effect being reduction in size of a tumor, reduction in metastasis, complete
remission, partial
remission, pathologic complete response, or stable disease. In some
embodiments, the PARP
inhibitor is a benzamide or a metabolite thereof. In some embodiments, the
benzamide is 4-
iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the anti-
tumor agent is
an antitumor alkylating agent, antitumor antimetabolite, antitumor
antibiotics, plant-derived
CA 02725026 2010-12-10
antitumor agent, antitumor platinum complex, antitumor camptothecin
derivative, antitumor
tyrosine kinase inhibitor, monoclonal antibody, interferon, biological
response modifier,
hormonal anti-tumor agent, anti-tumor viral agent, angiogenesis inhibitor,
differentiating
agent, or other agent that exhibits anti-tumor activities, or a
pharmaceutically acceptable salt
thereof. In some embodiments, the platinum complex is selected from the group
consisting
of cisplatin, carboplatin, oxaplatin and oxaliplatin. In some embodiments, the
platinum
complex is carboplatin. In some embodiments, the taxane is paclitaxel or
docetaxel. In some
embodiments, the taxane is paclitaxel. In some embodiments, the anti-tumor
agent is an anti-
angiogenic agent, such as Avastin or a receptor tyrosine kinase inhibitor
including but not
limited to Sutent, Nexavar, Recentin, ABT-869, and Axitinib. In some
embodiments, the anti-
tumor agent is a topoisomerase inhibitor including but not limited to
irinotecan, topotecan, or
camptothecin. In some embodiments, the anti-tumor agent is a taxane including
but not
limited to paclitaxel, docetaxel and Abraxane. In some embodiments, the anti-
tumor agent is
an agent targeting Her-2, e.g., Herceptin or lapatinib. In some embodiments,
the anti-tumor
agent is a hormone analog, for example, progesterone. In some embodiments, the
anti-tumor
agent is tamoxifen, a steroidal aromatase inhibitor, a non-steroidal aromatase
inhibitor, or
Fulvestrant. In some embodiments, the anti-tumor agent is an agent targeting a
growth factor
receptor. In some embodiments, such agent is an inhibitor of epidermal growth
factor
receptor (EGFR) including but not limited to Cetuximab and Panitumimab. In
some
embodiments, the agent targeting a growth factor receptor is an inhibitor of
insulin-like
growth factor 1 (IGF-1) receptor (IGF1R) such as CP-751871. In some
embodiments, the
cancer is a uterine cancer. In some embodiments, the cancer is advanced
uterine
carcinosarcoma, persistent uterine carcinosarcoma or recurrent uterine
carcinosarcoma. In
some embodiments, the cancer is endometrial cancer. In some embodiments, the
cancer is
ovarian cancer. In some embodiments, the cancer is a metastatic ovarian cancer
or uterine
cancer. In some embodiments, the method comprises selecting a treatment cycle
of at least 11
days and: (a) on day 1 of the cycle, administering to the patient about 10-200
mg/m2 of
paclitaxel; (b) on day 1 of the cycle, administering to the patient about 10-
400 mg/m2
carboplatin; and (c) on day 1 and twice weekly throughout the cycle,
administering to the
patient about 1-100 mg/kg of 4-iodo-3-nitrobenzamide or a molar equivalent of
a metabolite
thereof.
[0064] In some embodiments, the disclosure provides a method of treating
uterine cancer,
endometrial cancer, or ovarian cancer in a patient, comprising: (a) obtaining
a sample from
the patient; (b) testing the sample to determine a level of PARP expression in
the sample; (c)
21
CA 02725026 2010-12-10
determining whether the PARP expression exceeds a predetermined level, and if
so,
administering to the patient at least one taxane, at least one platinum
complex and at least one
PARP inhibitor. In some embodiments, the method further comprises optionally
selecting a
different treatment option if the PARP expression in the sample does not
exceed the
predetermined level. In some embodiments, the method optionally further
comprises
selecting a different treatment option if the PARP expression in the sample
does not exceed
the predetermined level. In some embodiments, the cancer is a uterine cancer.
In some
embodiments, the cancer is advanced uterine carcinosarcoma, persistent uterine
carcinosarcoma or recurrent uterine carcinosarcoma. In some embodiments, the
cancer is an
endometrial cancer. In some embodiments, the cancer is an ovarian cancer. In
some
embodiments, the cancer is a metastatic ovarian cancer. In some embodiments,
the taxane is
cisplatin, carboplatin, oxaplatin or oxaliplatin. In some embodiments, the
taxane is
paclitaxel. In some embodiments, the platinum complex is cisplatin or
carboplatin. In some
embodiments, the platinum complex is carboplatin. In some embodiments, the
PARP
inhibitor is a benzamide or a metabolite thereof. In some embodiments, the
PARP inhibitor
is 4-iodo-3-nitrobenzamide. In some embodiments, the sample is a tissue sample
or a bodily
fluid sample.
[0065] In some embodiments, the present disclosure provides a method of
treating uterine
cancer, endometrial cancer, or ovarian cancer in a patient, comprising during
a 21 day
treatment cycle: (a) on day 1 of the cycle, administering to the patient about
750 mg/m2 of
paclitaxel; (b) on day 1 of the cycle, administering to the patient about 10-
400 mg/m2 of
carboplatin; and (c) on day 1 of the cycle, and twice weekly thereafter,
administering to the
patient about 1-100 mg/kg of 4-iodo-3-nitrobenzamide. In some embodiments, the
paclitaxel
is administered as an intravenous infusion. In some embodiments, the
carboplatin is
administered as an intravenous infusion. In some embodiments, the 4-iodo-3-
nitrobenzamide
is administered orally or as a parenteral injection or infusion, or
inhalation. In some
embodiments, the cancer is a uterine cancer selected from advanced uterine
carcinosarcoma,
persistent uterine carcinosarcoma and recurrent uterine carcinosarcoma. In
some
embodiments, the cancer is ovarian cancer.
[0066] Some embodiments described herein provide a method of treating uterine
cancer,
endometrial cancer, or ovarian cancer in a patient, comprising: (a)
establishing a treatment
cycle of about 10 to about 30 days in length; (b) on from 1 to 5 separate days
of the cycle,
administering to the patient about 100 to about 2000 mg/m2 of paclitaxel by
intravenous
infusion over about 10 to about 300 minutes; (c) on from 1 to 5 separate days
of the cycle,
22
CA 02725026 2010-12-10
administering to the patient about 10-400 mg/m2 of carboplatin by intravenous
infusion over
about 10 to about 300 minutes; and (d) on from 1 to 10 separate days of the
cycle,
administering to the patient about 1 mg/kg to about 8 mg/kg of 4-iodo-3-
nitrobenzamide over
about 10 to about 300 minutes.
[0067] Some embodiments described herein provide a method of treating uterine
cancer
in a patient in need of such treatment, comprising: (a) testing a uterine
tumor sample from
the patient to determine at least one of the following: (i) whether the
uterine cancer is
advanced; (ii) whether the uterine cancer is persistent; (iii) whether the
uterine cancer is
recurrent; (b) if the testing indicates that the uterine cancer is advance,
persistent or recurrent,
treating the patient with a combination of therapeutic agents, wherein the
therapeutic agents
include at least one anti-tumor agent and at least one PARP inhibitor. In some
embodiments,
the at least one therapeutic effect is obtained, said at least one therapeutic
effect being
reduction in size of a uterine tumor, reduction in metastasis, complete
remission, partial
remission, pathologic complete response, or stable disease. In some
embodiments, the PARP
inhibitor is a benzamide or a metabolite thereof. In some embodiments, the
benzamide is 4-
iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the
platinum complex
is selected from the group consisting of cisplatin, carboplatin, oxaplatin and
oxaliplatin. In
some embodiments, the platinum complex is carboplatin. In some embodiments,
the taxane
is paclitaxel or docetaxel. In some embodiments, the taxane is paclitaxel. In
some
embodiments, the cancer is an advanced carcinosarcoma, a persistent
carcinosarcoma or a
recurrent carcinosarcoma. In some embodiments, the cancer is an endometrial
cancer.
[0068] In some embodiments, the method comprises treating a patient with at
least three
chemically distinct substances, one of which is a taxane (e.g., paclitaxel or
docetaxel), one of
which is a platinum-containing complex (e.g., cisplatin or carboplatin or
cisplatin) and one of
which is a PARP inhibitor (e.g., 4-iodo-3-nitrobenzamide or a metabolite
thereof). In some
embodiments, one or more of these substances may be capable of being present
in a variety
of physical forms-e.g., free base, salts (especially pharmaceutically
acceptable salts),
hydrates, polymorphs, solvates, or metabolites, etc. Unless otherwise
qualified herein, use of
a chemical name is intended to encompass all physical forms of the named
chemical. For
example, recitation of 4-iodo-3-nitrobenzamide, without further qualification,
is intended to
generically encompass the free base as well as all pharmaceutically acceptable
salts,
polymorphs, hydrates, and metabolites thereof. Where it is intended to limit
the disclosure or
claims to a particular physical form of a compound, this will be clear from
the context of the
passage or claim in which the reference to the compound appears.
23
CA 02725026 2010-12-10
Anti-tumor agents
[0069] Anti-tumor agents that may be used in the present invention include but
are not
limited to antitumor alkylating agents, antitumor antimetabolites, antitumor
antibiotics, plant-
derived antitumor agents, antitumor platinum-complex compounds, antitumor
camptothecin
derivatives, antitumor tyrosine kinase inhibitors, anti-tumor viral agent,
monoclonal
antibodies, interferons, biological response modifiers, and other agents that
exhibit anti-tumor
activities, or a pharmaceutically acceptable salt thereof.
[0070] In some embodiments, the anti-tumor agent is an alkylating agent. The
term
"alkylating agent" herein generally refers to an agent giving an alkyl group
in the alkylation
reaction in which a hydrogen atom of an organic compound is substituted with
an alkyl
group. Examples of anti-tumor alkylating agents include but are not limited to
nitrogen
mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan,
mitobronitol,
carboquone, thiotepa, ranimustine, nimustine, temozolomide or carmustine.
[0071] In some embodiments, the anti-tumor agent is an antimetabolite. The
term
"antimetabolite" used herein includes, in a broad sense, substances which
disturb normal
metabolism and substances which inhibit the electron transfer system to
prevent the
production of energy-rich intermediates, due to their structural or functional
similarities to
metabolites that are important for living organisms (such as vitamins,
coenzymes, amino
acids and saccharides). Examples of antimetabolites that have anti-tumor
activities include
but are not limited to methotrexate, 6-mercaptopurine riboside,
mercaptopurine, 5-
fluorouracil, tegafur, doxifluridine, carmofur, cytarabine, cytarabine
ocfosfate, enocitabine,
S-1, gemcitabine, fludarabine or pemetrexed disodium, and preferred are 5-
fluorouracil, S-1,
gemcitabine and the like.
[0072] In some embodiments, the anti-tumor agent is an antitumor antibiotic.
Examples
of antitumor antibiotics include but are not limited to actinomycin D,
doxorubicin,
daunorubicin, neocarzinostatin, bleomycin, peplomycin, mitomycin C,
aclarubicin,
pirarubicin, epirubicin, zinostatin stimalamer, idarubicin, sirolimus or
valrubicin.
In some embodiments, the anti-tumor agent is a plant-derived antitumor agent.
Examples of
plant-derived antitumor agents include but are not limited to vincristine,
vinblastine,
vindesine, etoposide, sobuzoxane, docetaxel, paclitaxel and vinorelbine, and
preferred are
docetaxel and paclitaxel.
[0073] In some embodiments, the anti-tumor agent is a camptothecin derivative
that
exhibits anti-tumor activities. Examples of anti-tumor camptothecin
derivatives include but
are not limited to camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan
or 9-
24
CA 02725026 2010-12-10
aminocamptothecin, with camptothecin, topotecan and irinotecan being
preferred. Further,
irinotecan is metabolized in vivo and exhibits antitumor effect as SN-38. The
action
mechanism and the activity of the camptothecin derivatives are believed to be
virtually the
same as those of camptothecin (e.g., Nitta, et al., Gan to Kagaku Ryoho, 14,
850-857 (1987)).
[0074] In some embodiments, the anti-tumor agent is an organoplatinum compound
or a
platinum coordination compound having antitumor activity. The terms
"organoplatinum
compound," "platinum compound," or "platinum complex" and the like as used
herein refer
to a platinum-containing compound which provides platinum in ion form.
Preferred
organoplatinum compounds include but are not limited to cisplatin; cis-
diamminediaquoplatinum (11)-ion; chloro(diethylenetriamine)-platinum (II)
chloride;
dichloro(ethylenediamine)-platinum (II); diammine(1,1-
cyclobutanedicarboxylato) platinum
(II) (carboplatin); spiroplatin; iproplatin; diammine(2-ethylmalonato)platinum
(II);
ethylenediaminemalonatoplatinum (II); aqua(1,2-
diaminodicyclohexane)sulfatoplatinum (II);
aqua(1,2-diaminodicyclohexane)malonatoplatinum (II); (1,2-
diaminocyclohexane)malonatoplatinum (II); (4-carboxyphthalato)(1,2-
diaminocyclohexane)
platinum (II); (1,2-diaminocyclohexane)-(isocitrato)platinum (II); (1,2-
diaminocyclohexane)oxalatoplatinum (II); ormaplatin; tetraplatin; carboplatin,
nedaplatin and
oxaliplatin, and preferred is carboplatin or oxaliplatin. Further, other
antitumor
organoplatinum compounds mentioned in the specification are known and are
commercially
available and/or producible by a person having ordinary skill in the art by
conventional
techniques.
[0075] In some embodiments, the anti-tumor agent is an antitumor tyrosine
kinase
inhibitor. The term "tyrosine kinase inhibitor" herein refers to a chemical
substance inhibiting
"tyrosine kinase" which transfers a k-phosphate group of ATP to a hydroxyl
group of a
specific tyrosine in protein. Examples of anti-tumor tyrosine kinase
inhibitors include but are
not limited to gefitinib, imatinib, erlotinib, Sutent, Nexavar, Recentin, ABT-
869, and
Axitinib.
[0076] In some embodiments, the anti-tumor agent is an antibody or a binding
portion of
an antibody that exhibits anti-tumor activity. In some embodiments, the anti-
tumor agent is a
monoclonal antibody. Examples thereof include but are not limited to
abciximab,
adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, daclizumab,
eculizumab,
efalizumab, ibritumomab, tiuxetan, infliximab, muromonab-CD3, natalizumab,
omalizumab,
palivizumab, panitumumab, ranibizumab, gemtuzumab ozogamicin, rituximab,
tositumomab,
trastuzumab, or any antibody fragments specific for antigens.
CA 02725026 2010-12-10
[0077] In some embodiments, the anti-tumor agent is an interferon. Such
interferon has
antitumor activity, and it is a glycoprotein which is produced and secreted by
most animal
cells upon viral infection. It has not only the effect of inhibiting viral
growth but also various
immune effector mechanisms including inhibition of growth of cells (in
particular, tumor
cells) and enhancement of the natural killer cell activity, thus being
designated as one type of
cytokine. Examples of anti-tumor interferons include but are not limited to
interferon a,
interferon a -2a, interferon a-2b, interferon 0, interferon y-la and
interferon y-nl.
[0078] In some embodiments, the anti-tumor agent is a biological response
modifier. It is
generally the generic term for substances or drugs for modifying the defense
mechanisms of
living organisms or biological responses such as survival, growth or
differentiation of tissue
cells in order to direct them to be useful for an individual against tumor,
infection or other
diseases. Examples of the biological response modifier include but are not
limited to krestin,
lentinan, sizofiran, picibanil and ubenimex.
[0079] In some embodiments, the anti-tumor agents include but are not limited
to
mitoxantrone, L-asparaginase, procarbazine, dacarbazine, hydroxycarbamide,
pentostatin,
tretinoin, alefacept, darbepoetin alfa, anastrozole, exemestane, bicalutamide,
leuprorelin,
flutamide, fulvestrant, pegaptanib octasodium, denileukin diftitox,
aldesleukin, thyrotropin
alfa, arsenic trioxide, bortezomib, capecitabine, and goserelin.
[0080] The above-described terms "antitumor alkylating agent", "antitumor
antimetabolite", "antitumor antibiotic", "plant-derived antitumor agent",
"antitumor platinum
coordination compound", "antitumor camptothecin derivative", "antitumor
tyrosine kinase
inhibitor", "monoclonal antibody", "interferon", "biological response
modifier" and "other
antitumor agent" are all known and are either commercially available or
producible by a
person skilled in the art by methods known per se or by well-known or
conventional methods.
The process for preparation of gefitinib is described, for example, in U.S.
Pat. No. 5,770,599;
the process for preparation of cetuximab is described, for example, in WO
96/40210; the
process for preparation of bevacizumab is described, for example, in WO
94/10202; the
process for preparation of oxaliplatin is described, for example, in U.S. Pat.
Nos. 5,420,319
and 5,959,133; the process for preparation of gemcitabine is described, for
example, in U.S.
Pat. Nos. 5,434,254 and 5,223,608; and the process for preparation of
camptothecin is
described in U.S. Pat. Nos. 5,162,532, 5,247,089, 5,191,082, 5,200,524,
5,243,050 and
5,321,140; the process for preparation of irinotecan is described, for
example, in U.S. Pat.
No. 4,604,463; the process for preparation of topotecan is described, for
example, in U.S. Pat.
No. 5,734,056; the process for preparation of temozolomide is described, for
example, in JP-
26
CA 02725026 2010-12-10
B No. 4-5029; and the process for preparation of rituximab is described, for
example, in JP-
W No. 2-503143.
[0081] The above-mentioned antitumor alkylating agents are commercially
available, as
exemplified by the following: nitrogen mustard N-oxide from Mitsubishi Pharma
Corp. as
Nitrorin (tradename); cyclophosphamide from Shionogi & Co., Ltd. as Endoxan
(tradename);
ifosfamide from Shionogi & Co., Ltd. as Ifomide (tradename); melphalan from
GlaxoSmithKline Corp. as Alkeran (tradename); busulfan from Takeda
Pharmaceutical Co.,
Ltd. as Mablin (tradename); mitobronitol from Kyorin Pharmaceutical Co., Ltd.
as Myebrol
(tradename); carboquone from Sankyo Co., Ltd. as Esquinon (tradename);
thiotepa from
Sumitomo Pharmaceutical Co., Ltd. as Tespamin (tradename); ranimustine from
Mitsubishi
Pharma Corp. as Cymerin (tradename); nimustine from Sankyo Co., Ltd. as Nidran
(tradename); temozolomide from Schering Corp. as Temodar (tradename); and
carmustine
from Guilford Pharmaceuticals Inc. as Gliadel Wafer (tradename).
[0082] The above-mentioned antitumor antimetabolites are commercially
available, as
exemplified by the following: methotrexate from Takeda Pharmaceutical Co.,
Ltd. as
Methotrexate (tradename); 6-mercaptopurine riboside from Aventis Corp. as
Thioinosine
(tradename); mercaptopurine from Takeda Pharmaceutical Co., Ltd. as Leukerin
(tradename);
5-fluorouracil from Kyowa Hakko Kogyo Co., Ltd. as 5-FU (tradename); tegafur
from Taiho
Pharmaceutical Co., Ltd. as Futraful (tradename); doxyfluridine from Nippon
Roche Co.,
Ltd. as Furutulon (tradename); carmofur from Yamanouchi Pharmaceutical Co.,
Ltd. as
Yamafur (tradename); cytarabine from Nippon Shinyaku Co., Ltd. as Cylocide
(tradename);
cytarabine ocfosfate from Nippon Kayaku Co., Ltd. as Strasid(tradename);
enocitabine from
Asahi Kasei Corp. as Sanrabin (tradename); S-1 from Taiho Pharmaceutical Co.,
Ltd. as TS-1
(tradename); gemcitabine from Eli Lilly & Co. as Gemzar (tradename);
fludarabine from
Nippon Schering Co., Ltd. as Fludara (tradename); and pemetrexed disodium from
Eli Lilly
& Co. as Alimta (tradename).
[0083] The above-mentioned antitumor antibiotics are commercially available,
as
exemplified by the following: actinomycin D from Banyu Pharmaceutical Co.,
Ltd. as
Cosmegen (tradename); doxorubicin from Kyowa Hakko Kogyo Co., Ltd. as adriacin
(tradename); daunorubicin from Meiji Seika Kaisha Ltd. as Daunomycin;
neocarzinostatin
from Yamanouchi Pharmaceutical Co., Ltd. as Neocarzinostatin (tradename);
bleomycin
from Nippon Kayaku Co.; Ltd. as Bleo (tradename); pepromycin from Nippon
Kayaku Co,
Ltd. as Pepro (tradename); mitomycin C from Kyowa Hakko Kogyo Co., Ltd. as
Mitomycin
(tradename); aclarubicin from Yamanouchi Pharmaceutical Co., Ltd, as Aclacinon
27
CA 02725026 2010-12-10
(tradename); pirarubicin from Nippon Kayaku Co., Ltd. as Pinorubicin
(tradename);
epirubicin from Pharmacia Corp. as Pharmorubicin (tradename); zinostatin
stimalamer from
Yamanouchi Pharmaceutical Co., Ltd. as Smancs (tradename); idarubicin from
Pharmacia
Corp. as Idamycin (tradename); sirolimus from Wyeth Corp. as Rapamune
(tradename); and
valrubicin from Anthra Pharmaceuticals Inc. as Valstar (tradename).
[0084] The above-mentioned plant-derived antitumor agents are commercially
available,
as exemplified by the following: vincristine from Shionogi & Co., Ltd. as
Oncovin
(tradename); vinblastine from Kyorin Pharmaceutical Co., Ltd. as Vinblastine
(tradename);
vindesine from Shionogi & Co., Ltd. as Fildesin (tradename); etoposide from
Nippon Kayaku
Co., Ltd. as Lastet (tradename); sobuzoxane from Zenyaku Kogyo Co., Ltd. as
Perazolin
(tradename); docetaxel from Aventis Corp. as Taxsotere (tradename); paclitaxel
from Bristol-
Myers Squibb Co. as Taxol (tradename); and vinorelbine from Kyowa Hakko Kogyo
Co.,
Ltd. as Navelbine (tradename).
[0085] The above-mentioned antitumor platinum coordination compounds are
commercially available, as exemplified by the following: cisplatin from Nippon
Kayaku Co.,
Ltd. as Randa (tradename); carboplatin from Bristol-Myers Squibb Co. as
Paraplatin
(tradename); nedaplatin from Shionogi & Co., Ltd. as Aqupla (tradename); and
oxaliplatin
from Sanofi-Synthelabo Co. as Eloxatin (tradename).
[0086] The above-mentioned antitumor camptothecin derivatives are commercially
available, as exemplified by the following: irinotecan from Yakult Honsha Co.,
Ltd. as
Campto (tradename); topotecan from GlaxoSmithKline Corp. as Hycamtin
(tradename); and
camptothecin from Aldrich Chemical Co., Inc., U.S.A.
[0087] The above-mentioned antitumor tyrosine kinase inhibitors are
commercially
available, as exemplified by the following: gefitinib from AstraZeneca Corp.
as Iressa
(tradename); imatinib from Novartis AG as Gleevec (tradename); and erlotinib
from OSI
Pharmaceuticals Inc. as Tarceva (tradename).
[0088] The above-mentioned monoclonal antibodies are commercially available,
as
exemplified by the following: cetuximab from Bristol-Myers Squibb Co. as
Erbitux
(tradename); bevacizumab from Genentech, Inc. as Avastin (tradename);
rituximab from
Biogen Idec Inc. as Rituxan (tradename); alemtuzumab from Berlex Inc. as
Campath
(tradename); and trastuzumab from Chugai Pharmaceutical Co., Ltd. as Herceptin
(tradename).
[0089] The above-mentioned interferons are commercially available, as
exemplified by
the following: interferon a from Sumitomo Pharmaceutical Co., Ltd. as
Sumiferon
28
CA 02725026 2010-12-10
(tradename); interferon a-2a from Takeda Pharmaceutical Co., Ltd. as Canferon-
A
(tradename); interferon a-2b from Schering-Plough Corp. as Intron A
(tradename); interferon
(3 from Mochida Pharmaceutical Co., Ltd. as IFN.beta. (tradename); interferon
y-la from
Shionogi & Co., Ltd. as Imunomax-y (tradename); and interferon y-nl from
Otsuka
Pharmaceutical Co., Ltd. as Ogamma (tradename).
[0090] The above-mentioned biological response modifiers are commercially
available,
as exemplified by the following: krestin from Sankyo Co., Ltd. as krestin
(tradename);
lentinan from Aventis Corp. as Lentinan (tradename); sizofiran from Kaken
Seiyaku Co., Ltd.
as Sonifiran (tradename); picibanil from Chugai Pharmaceutical Co., Ltd. as
Picibanil
(tradename); and ubenimex from Nippon Kayaku Co., Ltd. as Bestatin
(tradename).
[0091] The above-mentioned other antitumor agents are commercially available,
as
exemplified by the following: mitoxantrone from Wyeth Lederle Japan, Ltd. as
Novantrone
(tradename); L-asparaginase from Kyowa Hakko Kogyo Co., Ltd. as Leunase
(tradename);
procarbazine from Nippon Roche Co., Ltd. as Natulan (tradename); dacarbazine
from Kyowa
Hakko Kogyo Co., Ltd. as Dacarbazine (tradename); hydroxycarbamide from
Bristol-Myers
Squibb Co. as Hydrea (tradename); pentostatin from Kagaku Oyobi Kessei Ryoho
Kenkyusho as Coforin (tradename); tretinoin from Nippon Roche Co., Ltd. As
Vesanoid
(tradename); alefacept from Biogen Idec Inc. as Amevive (tradename);
darbepoetin alfa from
Amgen Inc. as Aranesp (tradename); anastrozole from AstraZeneca Corp. as
Arimidex
(tradename); exemestane from Pfizer Inc. as Aromasin (tradename); bicalutamide
from
AstraZeneca Corp. as Casodex (tradename); leuprorelin from Takeda
Pharmaceutical Co.,
Ltd. as Leuplin (tradename); flutamide from Schering-Plough Corp. as Eulexin
(tradename);
fulvestrant from AstraZeneca Corp. as Faslodex (tradename); pegaptanib
octasodium from
Gilead Sciences, Inc. as Macugen (tradename); denileukin diftitox from Ligand
Pharmaceuticals Inc. as Ontak (tradename); aldesleukin from Chiron Corp. as
Proleukin
(tradename); thyrotropin alfa from Genzyme Corp. as Thyrogen (tradename);
arsenic trioxide
from Cell Therapeutics, Inc. as Trisenox (tradename); bortezomib from
Millennium
Pharmaceuticals, Inc. as Velcade (tradename); capecitabine from Hoffmann-La
Roche, Ltd.
as Xeloda (tradename); and goserelin from AstraZeneca Corp. as Zoladex
(tradename). The
term "antitumor agent" as used in the specification includes the above-
described antitumor
alkylating agent, antitumor antimetabolite, antitumor antibiotic, plant-
derived antitumor
agent, antitumor platinum coordination compound, antitumor camptothecin
derivative,
antitumor tyrosine kinase inhibitor, monoclonal antibody, interferon,
biological response
modifier, and other antitumor agents.
29
CA 02725026 2010-12-10
[0092] Other anti-tumor agents or anti-neoplastic agents can be used in
combination with
benzopyrone compounds. Such suitable anti-tumor agents or anti-neoplastic
agents include,
but are not limited to, 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-
Azacitidine,
5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane,
Accutane,
Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin,
Alemtuzumab,
ALIMTA, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic Acid, Alpha
Interferon,
Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide,
Anandron,
Anastrozole, Arabinosylcytosine, Ara-C, Aranesp, Aredia, Arimidex, Aromasin,
Arranon,
Arsenic Trioxide, Asparaginase, ATRA, Avastin, Azacitidine, BCG, BCNU,
Bendamustine,
Bevacizumab, Bexarotene, BEXXAR, Bicalutamide, BiCNU, Blenoxane, Bleomycin,
Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar,
Camptothecin- 11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine
Wafer,
Casodex, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine, Cetuximab,
Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen,
CPT-11,
Cyclophosphamide, Cytadren, Cytarabine, Cytarabine Liposomal, Cytosar-U,
Cytoxan,
Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin,
Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome,
Decadron, Decitabine, Delta-Cortef, Deltasone, Denileukin Diftitox, DepoCytTM
Dexamethasone, Dexamethasone Acetate, Dexamethasone Sodium Phosphate,
Dexasone,
Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin
Liposomal,
DroxiaTM, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin,
Elspar, Emcyt,
Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-asparaginase,
Estramustine, Ethyol,
Etopophos, Etoposide, Etoposide Phosphate, Eulexin, Evista, Exemestane,
Fareston,
Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex,
Fluorouracil,
Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR ,
Fulvestrant, G-
CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar & Gemzar Side
Effects -
Chemotherapy Drugs, Gleevec, Gliadel Wafer, GM-CSF, Goserelin, Granulocyte -
Colony
Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor,
Halotestm,
Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea,
Hydrocort
Acetate, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone
Sodium
Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab
Tiuxetan,
Idamycin, Idarubicin, Ifex , IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib
mesylate, Imidazole
Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin -
2,
Interleukin- 11, Intron A (interferon alfa-2b), Iressa, Irinotecan,
Isotretinoin, Ixabepilone,
CA 02725026 2010-12-10
Ixempra, Kidrolase (t), Lanacort, Lapatinib, L-asparaginase, LCR,
Lenalidomide, Letrozole,
Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal
Ara-C,
Liquid Pred, Lomustine, L-PAM, L-Sarcolysin, Lupron, Lupron Depot, Matulane,
Maxidex,
Mechlorethamine, Mechlorethamine Hydrochloride, Medralone, Medrol, Megace,
Megestrol,
Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate,
Methotrexate
Sodium, Methylprednisolone, Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone,
M-
Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Mylocel,
Mylotarg,
Navelbine, Nelarabine, Neosar, Neulasta, Neumega, Neupogen, Nexavar,
Nilandron,
Nilutamide, Nipent, Nitrogen Mustard, Novaldex, Novantrone, Octreotide,
Octreotide
acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone,
Oxaliplatin,
Paclitaxel, Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin,
Paraplatin,
Pediapred, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-
asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard, Platinol,
Platinol-AQ,
Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin,
Prolifeprospan 20
with Carmustine Implant, Purinethol, Raloxifene, Revlimid, Rheumatrex,
Rituxan,
Rituximab, Roferon-A (Interferon Alfa-2a), Rubex, Rubidomycin hydrochloride,
Sandostatin,
Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, Sorafenib, SPRYCEL,
STI-571,
Streptozocin, SU11248, Sunitinib, Sutent, Tamoxifen, Tarceva, Targretin,
Taxol, Taxotere,
Temodar, Temozolomide, Temsirolimus, Teniposide, TESPA, Thalidomide, Thalomid,
TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex,
Thiotepa,
TICE, Toposar, Topotecan, Toremifene, Torisel, Tositumomab, Trastuzumab,
Tretinoin,
TrexallTM, Trisenox, TSPA, TYKERB, VCR, Vectibix, Vectibix, Velban, Velcade,
VePesid,
Vesanoid, Viadur, Vidaza, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,
Vincristine,
Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon,
Xeloda,
Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zolinza, Zometa.
Antimetabolites
[0093] Antimetabolites are drugs that interfere with normal cellular metabolic
processes.
Since cancer cells are rapidly replicating, interference with cellular
metabolism affects cancer
cells to a greater extent than host cells. Gemcitabine (4-amino-1-[3,3-
difluoro-4-hydroxy-5-
(hydroxymethyl) tetrahydrofuran-2-yl]-1H-pyrimidin-2-one; marketed as GEMZAR
by Eli
Lilly and Company) is a nucleoside analog, which interferes with cellular
division by
blocking DNA synthesis, thus resulting in cell death, apparently through an
apoptotic
mechanism. The dosage of gemcitabine may be adjusted to the particular
patient. In adults,
31
CA 02725026 2010-12-10
the dosage of gemcitabine, when used in combination with a platinum agent and
a PARP
inhibitor, will be in the range of about 100 mg/m2 to about 5000 mg/m2, in the
range of about
100 mg/m2 to about 2000 mg/m2, in the range of about 750 to about 1500 mg/m2,
about 900
to about 1400 mg/m2 or about 1250 mg/m2. The dimensions mg/m2 refer to the
amount of
gemcitabine in milligrams (mg) per unit surface area of the patient in square
meters (m).
Gemcitabine may be administered by intravenous (IV) infusion, e.g., over a
period of about
to about 300 minutes, about 15 to about 180 minutes, about 20 to about 60
minutes or
about 10 minutes. The term "about" in this context indicates the normal usage
of
approximately; and in some embodiments indicates a tolerance of 10% or 5%.
Taxanes
[0094] Taxanes are drugs that are derived from the twigs, needles and bark of
Pacific yew
tress, Taxus brevifolia. In particular paclitaxel may be derived from 10-
deacetylbaccatin
through known synthetic methods. Taxanes such as paclitaxel and its derivative
docetaxel
have demonstrated antitumor activity in a variety of tumor types. The taxanes
interfere with
normal function of microtubule growth by hyperstabilizing their structure,
thereby destroying
the cell's ability to use its cytoskeleton in a normal manner. Specifically,
the taxanes bind to
the (3 subunit of tubulin, which is the building block of microtubules. The
resulting
taxane/tubulin complex cannot disassemble, which results in aberrant cell
function and
eventual cell death. Paclitaxel induces programmed cell death (apoptosis) in
cancer cells by
binding to an apoptosis-inhibiting protein called Bcl-2 (B-cell leukemia 2),
thereby
preventing Bcl-2 from inhibiting apoptosis. Thus paclitaxel has proven to be
an effective
treatment for various cancers, as it down-regulates cell division by
interrupting normal
cytoskeletal rearrangement during cell division and it induces apoptosis via
the anti-Bcl-2
mechanism.
[0095] The dosage of paclitaxel may vary depending upon the height, weight,
physical
condition, tumor size and progression state, etc. In some embodiments, the
dosage of
paclitaxel will be in the range of about 10 to about 2000 mg/m2, about 10 to
about 200 mg/m2
or about 100 to about 175 mg/m2. In some embodiments, the paclitaxel will be
administered
over a period of up to about 10 hours, up to about 8 hours or up to about 6
hours. The term
"about" in this context indicates the normal usage of approximately; and in
some
embodiments indicates a tolerance of 10% or 5%.
[0096] Examples of taxanes include but are not limited to docetaxel,
paclitaxel, and
Abraxane.
32
CA 02725026 2010-12-10
Platinum complexes
[0097] Platinum complexes are pharmaceutical compositions used to treat
cancer, which
contain at least one platinum center complexed with at least one organic
group. Carboplatin
((SP-4-2)-Diammine[ 1, 1 -cyclobutanedicarboxylato(2-)-O,O' platinum), like
cisplatin and
oxaliplatin, is a DNA alkylating agent. The dosage of a platinum compound,
e.g.,
carboplatin, is determined by calculating the area under the blood plasma
concentration
versus time curve (AUC) in mg/mL=minute by methods known to those skilled in
the cancer
chemotherapy art, taking into account the patient's renal activity estimated
by measuring
creatinine clearance or glomerular filtration rate. In some embodiments, the
dosage of
carboplatin for combination treatment along with an antimetabolite (e.g.,
gemcitabine) and a
PARP inhibitor (e.g., 4-iodo-3-nitrobenzamide) is calculated to provide an AUC
of about 0.1-
6 mg/ml-min, about 1-3 mg/ml-min, about 1.5 to about 2.5 mg/ml-min, about 1.75
to about
2.25 mg/ml-min or about 2 mg/ml=min. (AUC 2, for example, is shorthand for 2
mg/ml-minute). Alternatively, the dosage of platinum compound, e.g.,
carboplatin, is
calculated based on the patient's body surface area. In some embodiments, a
suitable dose of
carboplatin is about 10 to about 400 mg/m2, e.g., about 360 mg/m2. Platinum
complexes,
such as carboplatin, are normally administered intravenously (IV) over a
period of about 10
to about 300 minutes, about 30 to about 180 minutes, about 45 to about 120
minutes or about
60 minutes. In this context, the term "about" has its normal meaning of
approximately. In
some embodiments, about means 10% or 5%.
Topoisomerase inhibitors
[0098] In some embodiments, the methods of the invention may comprise
administering
to a patient with uterine cancer or ovarian cancer an effective amount of a
PARP inhibitor in
combination with a topoisomerase inhibitor, for example, irinotecan and
topotecan.
[0099] Topoisomerase inhibitors are agents designed to interfere with the
action of
topoisomerase enzymes (topoisomerase I and II), which are enzymes that control
the changes
in DNA structurehttp://en.wikipedia.org/wiki/Topoisomerase_inhibitor -
cite_note-
urlDorlands_Medical_Dictionary:topoisomerase_inhibitor- I #cite_note-
urlDorlands_Medical_Dictionary:topoisomerase_inhibitor-1 by catalyzing the
breaking and
rejoining of the phosphodiester backbone of DNA strands during the normal cell
cycle.
Topoisomerases have become popular targets for cancer chemotherapy treatments.
It is
thought that topoisomerase inhibitors block the ligation step of the cell
cycle, generating
single and double stranded breaks that harm the integrity of the genome.
Introduction of these
33
CA 02725026 2010-12-10
breaks subsequently lead to apoptosis and cell death. Topoisomerase inhibitors
are often
divided according to which type of enzyme it inhibits. Topoisomerase I, the
type of
topoisomerase most often found in eukaryotes, is targeted by topotecan,
irinotecan, lurtotecan
and exatecan, each of which is commercially available. Topotecan is available
from
G1axoSmithKline under the trade name Hycamtim . Irinotecan is available from
Pfizer
under the trade name Camptosar . Lurtotecan may be obtained as a liposomal
formulation
from Gilead Sciences Inc. Topoisomerase inhibitors may be administered at an
effective
dose. In some embodiments an effective dose for treatment of a human will be
in the range
of about 0.01 to about 10 mg/m2/day. The treatment may be repeated on a daily,
bi-weekly,
semi-weekly, weekly, or monthly basis. In some embodiments, a treatment period
may be
followed by a rest period of from one day to several days, or from one to
several weeks. In
combination with a PARP-1 inhibitor, the PARP-1 inhibitor and the
topoisomerase inhibitor
may be dosed on the same day or may be dosed on separate days.
[0100] Compounds that target type II topoisomerase are split into two main
classes:
topoisomerase poisons, which target the topoisomerase-DNA complex, and
topoisomerase
inhibitors, which disrupt catalytic turnover. Topo II poisons include but are
not limited to
eukaryotic type II topoisomerase inhibitors (topo II): amsacrine, etoposide,
etoposide
phosphate, teniposide, amrubicin and doxorubicin. These drugs are anti-cancer
therapies.
Examples of topoisomerase inhibitors include ICRF-193. These inhibitors target
the N-
terminal ATPase domain of topo II and prevent topo II from turning over. The
structure of
this compound bound to the ATPase domain has been solved by Classen
(Proceedings of the
National Academy of Science, 2004) showing that the drug binds in a non-
competitive
manner and locks down the dimerization of the ATPase domain.
Anti-angiogenic agents
[0101] In some embodiments, the methods of the invention may comprise
administering
to a patient with uterine, endometrial, or ovarian cancer an effective amount
of a PARP
inhibitor in combination with an anti-angiogenic agent.
[0102] An angiogenesis inhibitor is a substance that inhibits angiogenesis
(the growth of
new blood vessels). Every solid tumor (in contrast to leukemia) needs to
generate blood
vessels to keep it alive once it reaches a certain size. Tumors can grow only
if they form new
blood vessels. Usually, blood vessels are not built elsewhere in an adult body
unless tissue
repair is actively in process. The angiostatic agent endostatin and related
chemicals can
suppress the building of blood vessels, preventing the cancer from growing
indefinitely. In
34
CA 02725026 2010-12-10
tests with patients, the tumor became inactive and stayed that way even after
the endostatin
treatment is finished. The treatment has very few side effects but appears to
have very limited
selectivity. Other angiostatic agents such as thalidomide and natural plant-
based substances
are being actively investigated.
[0103] Known inhibitors include the drug bevacizumab (Avastin), which binds
vascular
endothelial growth factor (VEGF), inhibiting its binding to the receptors that
promote
angiogenesis. Other anti-angiogenic agents include but are not limited to
carboxyamidotriazole, TNF-470, CM101, IFN-alpha, IL-12, platelet factor-4,
suramin,
SU5416, thrombospondin, angiostatic steroids + heparin, cartilage-derived
angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors, angiostatin,
endostatin, 2-
methoxyestradiol, tecogalan, thrombospondin, prolactin, av(33 inhibitors and
linomide.
Her-2 targeted therapy
[0104] In some embodiments, the methods of the invention may comprise
administering
to a patient with HER2-positive uterine, endometrial, or ovarian cancer an
effective amount
of a PARP inhibitor in combination with Herceptin.
[0105] Her-2 overexpression has been found in ovarian carcinomas and HER2
overexpression and amplification is associated with advanced ovarian cancer
(AOC)
(Hellstrom et al., Cancer Research 61, 2420-2423, March 15, 2001).
Overexpression of
HER-2/neu in endometrial cancer is associated with advanced stage disease
(Berchuck A, et
al., Am J Obstet Gynecol. 1991 Jan;164(1 Pt 1):15-21). Herceptin may be used
for the
adjuvant treatment of HER2-overexpressing, uterine, endometrial, or ovarian
cancers.
Herceptin can be used several different ways: as part of a treatment regimen
including
doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel; with
docetaxel and
carboplatin; or as a single agent following multi-modality anthracycline-based
therapy.
Herceptin in combination with paclitaxel is approved for the first-line
treatment of HER2-
overexpressing uterine, endometrial, or ovarian cancers. Herceptin as a single
agent is
approved for treatment of HER2-overexpressing uterine, endometrial, or ovarian
cancer in
patients who have received one or more chemotherapy regimens for metastatic
disease.
[0106] Lapatinib or lapatinib ditosylate is an orally active chemotherapeutic
drug
treatment for solid tumours such as breast cancer. During development it was
known as small
molecule GW572016. Lapatinib may stop the growth of tumor cells by blocking
some of the
enzymes needed for cell growth. Drugs used in chemotherapy, such as topotecan,
work in
different ways to stop the growth of tumor cells, either by killing the cells
or by preventing
CA 02725026 2010-12-10
them from dividing. Giving lapatinib together with topotecan may have enhanced
anti-tumor
efficacy.
Hormone therapy
[0107] In some embodiments, the methods of the invention may comprise
administering
to a patient with uterine, endometrial, or ovarian cancer an effective amount
of a PARP
inhibitor in combination with hormone therapy.
[0108] Treatment for uterine cancer depends on the stage of the disease and
the overall
health of the patient. Removal of the tumor (surgical resection) is the
primary treatment.
Radiation therapy, hormone therapy, and/or chemotherapy may be used as
adjuvant treatment
(i.e., in addition to surgery) in patients with metastatic or recurrent
disease.
[0109] Hormone therapy is used to treat metastatic or recurrent endometrial
cancer. It
also may be used to treat patients who are unable to undergo surgery or
radiation. Prior to
treatment, a hormone receptor test may be performed to determine if the
endometrial tissue
contains these proteins. Hormone therapy usually involves a synthetic type of
progesterone in
pill form. Estrogen can cause the growth of ovarian epithelial cancer cells.
Thus, hormone
therapy may be used to treat ovarian cancer.
Tamoxifen-Hormone antagonist
[0110] Tamoxifen (marketed as Nolvadex) slows or stops the growth of cancer
cells
present in the body. Tamoxifen is a type of drug called a selective estrogen-
receptor
modulator (SERM). It functions as an anti-estrogen. As tamoxifen may have
stabilized
rapidly advancing recurrent ovarian cancer, its role in the primary treatment
of ovarian cancer
in combination with cytotoxic chemotherapy should be considered.
[0111] Steroidal and non-steroidal aromatase inhibitor. Aromatase inhibitors
(AI) are a
class of drugs used in the treatment of ovarian cancer in postmenopausal women
that block
the aromatase enzyme. Aromatase inhibitors lower the amount of estrogen in
post-
menopausal women who have hormone-receptor-positive ovarian cancer. With less
estrogen
in the body, the hormone receptors receive fewer growth signals, and cancer
growth can be
slowed down or stopped.
[0112] Aromatase inhibitor medications include Arimidex (chemical name:
anastrozole),
Aromasin (chemical name: exemestane), and Femara (chemical name: letrozole).
Each is
taken by pill once a day, for up to five years. But for women with advanced
(metastatic)
disease, the medicine is continued as long as it is working well.
36
CA 02725026 2010-12-10
[0113] AIs are categorized into two types: irreversible steroidal inhibitors
such as
exemestane that form a permanent bond with the aromatase enzyme complex; and
non-
steroidal inhibitors (such as anastrozole, letrozole) that inhibit the enzyme
by reversible
competition.
[0114] Fulvestrant, also known as ICI 182,780, and "Faslodex" is a drug
treatment of
hormone receptor-positive ovarian cancer in postmenopausal women with disease
progression following anti-estrogen therapy. Estrogen can cause the growth of
ovarian
epithelial cancer cells. Fulvestrant is an estrogen receptor antagonist with
no agonist effects,
which works both by down-regulating and by degrading the estrogen receptor. It
is
administered as a once-monthly injection.
Targeted therapy
[0115] In some embodiments, the methods of the invention may comprise
administering
to a patient with uterine, endometrial, or ovarian cancer an effective amount
of a PARP
inhibitor in combination with an inhibitor targeting a growth factor receptor
including but not
limited to epidermal growth factor receptor (EGFR) and insulin-like growth
factor 1 receptor
(IGF1R).
[0116] EGFR is overexpressed in the cells of certain types of human carcinomas
including but not limited to lung, breast, uterine, endometrial, and ovarian
cancers. EGFR
over-expression in ovarian cancer has been associated with poor prognosis. In
addition,
EGFR has been shown to be highly expressed in normal endometrium and
overexpressed in
endometrial cancer specimens, where it has been associated with a poor
prognosis. Increased
expression of EGFR may contribute to a drug resistant phenotype. The tyrosine
kinase
inhibitor ZD1839 (Iressa7M) has been studied as a single agent in a phase II
clinical trial (GOG
229C) of women with advanced endometrial cancer. Preliminary data analysis
indicates that
of 29 patients enrolled, 1 patient experienced a complete response and several
others had
stable disease at 6 months (Leslie, K.K.; et al., International Journal of
Gynecological
Cancer, Volume 15, Number 2, 2005, pp. 409-411(3). Examples of EGFR inhibitors
include
but are not limited to cetuximab, which is a chimeric monoclonal antibody
given by
intravenous injection for treatment of cancers including but not limited to
metastatic
colorectal cancer and head and neck cancer. Panitumimab is another example of
EGFR
inhibitor. It is a humanized monoclonal antibody against EGFR. Panitumimab has
been
shown to be beneficial and better than supportive care when used alone in
patients with
advanced colon cancer and is approved by the FDA for this use.
37
CA 02725026 2010-12-10
[0117] Activation of the type 1 insulin-like growth factor receptor (IGF1R)
promotes
proliferation and inhibits apoptosis in a variety of cell types. One example
of an IGF1R
inhibitor is CP-751871. CP-751871 is a human monoclonal antibody that
selectively binds to
IGF1R, preventing IGF1 from binding to the receptor and subsequent receptor
autophosphorylation. Inhibition of IGF1R autophosphorylation may result in a
reduction in
receptor expression on tumor cells that express IGF1R, a reduction in the anti-
apoptotic effect
of IGF, and inhibition of tumor growth. IGF1R is a receptor tyrosine kinase
expressed on
most tumor cells and is involved in mitogenesis, angiogenesis, and tumor cell
survival.
PI3K/mTOR pathway
[0118] Phosphatidylinositol-3-kinase (P13K) pathway deregulation is a common
event in
human cancer, either through inactivation of the tumor suppressor phosphatase
and tensin
homologue deleted from chromosome 10 or activating mutations of p110-a. These
hotspot
mutations result in oncogenic activity of the enzyme and contribute to
therapeutic resistance
to the anti-HER2 antibody trastuzumab. Akt and mTOR phosphorylation is also
frequently
detected in ovarian and endometrial cancer. The P13K pathway is, therefore, an
attractive
target for cancer therapy. NVP-BEZ235, a dual inhibitor of the P13K and the
downstream
mammalian target of rapamycin (mTOR) has been shown to have antiproliferative
and
antitumoral activity in cancer cells with both wild-type and mutated p110-a
(Violeta Serra, et
al., Cancer Research 68, 8022-8030, October 1, 2008).
Hsp90 inhibitors
[0119] These drugs target heat shock protein 90 (hsp90). Hsp90 is one of a
class of
chaperone proteins, whose normal job is to help other proteins acquire and
maintain the shape
required for those proteins to do their jobs. Chaperone proteins work by being
in physical
contact with other proteins. Hsp90 can also enable cancer cells to survive and
even thrive
despite genetic defects which would normally cause such cells to die. Thus,
blocking the
function of HSP90 and related chaperone proteins may cause cancer cells to
die, especially if
blocking chaperone function is combined with other strategies to block cancer
cell survival.
Tubulin inhibitors
[0120] Tubulins are the proteins that form microtubules, which are key
components of
the cellular cytoskeleton (structural network). Microtubules are necessary for
cell division
(mitosis), cell structure, transport, signaling and motility. Given their
primary role in mitosis,
microtubules have been an important target for anticancer drugs - often
referred to as
38
CA 02725026 2010-12-10
antimitotic drugs, tubulin inhibitors and microtubule targeting agents. These
compounds bind
to tubulin in microtubules and prevent cancer cell proliferation by
interfering with the
microtubule formation required for cell division. This interference blocks the
cell cycle
sequence, leading to apoptosis.
Apoptosis inhibitors
[0121] The inhibitors of apoptosis (IAP) are a family of functionally- and
structurally-
related proteins, originally characterized in Baculovirus, which serve as
endogenous
inhibitors of apoptosis. The human IAP family consists of at least 6 members,
and IAP
homologs have been identified in numerous organisms. 10058-F4 is a c-Myc
inhibitor that
induces cell-cycle arrest and apoptosis. It is a cell-permeable thiazolidinone
that specifically
inhibits the c-Myc-Max interaction and prevents transactivation of c-Myc
target gene
expression. 10058-F4 inhibits tumor cell growth in a c-Myc-dependent manner
both in vitro
and in vivo. BI-6C9 is a tBid inhibitor and antiapoptotic. GNF-2 belongs to a
new class of
Bcr-abl inhibitors. GNF-2 appears to bind to the myristoyl binding pocket, an
allosteric site
distant from the active site, stabilizing the inactive form of the kinase. It
inhibits Bcr-abl
phosphorylation with an IC50 of 267 nM, but does not inhibit a panel of 63
other kinases,
including native c-Abl, and shows complete lack of toxicity towards cells not
expressing Bcr-
Abl. GNF-2 shows great potential for a new class of inhibitor to study Bcr-abl
activity and to
treat resistant Chronic myelogenous leukemia (CML), which is caused the Bcr-
Abl
oncoprotein. Pifithrin-a is a reversible inhibitor of p53-mediated apoptosis
and p53-
dependent gene transcription such as cyclin G, p21/waft, and mdm2 expression.
Pifithrin-a
enhances cell survival after genotoxic stress such as UV irradiation and
treatment with
cytotoxic compounds including doxorubicin, etopoxide, paclitaxel,,and cytosine-
f -D-
arabinofuranoside. Pifithrin-a protects mice from lethal whole body y-
irradiation without an
increase in cancer incidence.
PARP Inhibitors
[0122] In some embodiments, the present invention provides a method of
treating uterine
cancer or ovarian cancer by administering to a subject in need thereof at
least one PARP
inhibitor. In other embodiments, the present invention provides a method of
treating uterine
cancer or ovarian cancer by administering to a subject in need thereof at
least one PARP
inhibitor in combination with at least one anti-tumor agent described herein.
[0123] Not intending to be limited to any particular mechanism of action, the
compounds
described herein are believed to have anti-cancer properties due to the
modulation of activity
39
CA 02725026 2010-12-10
= of a poly (ADP-ribose) polymerase (PARP). This mechanism of action is
related to the
ability of PARP inhibitors to bind PARP and decrease its activity. PARP
catalyzes the
conversion of 0-nicotinamide adenine dinucleotide (NAD+) into nicotinamide and
poly-
ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to
regulation of
transcription, cell proliferation, genomic stability, and carcinogenesis
(Bouchard V.J. et al.,
Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9);
Herceg
Z.; Wang Z.-Q. Mutation Research/ Fundamental and Molecular Mechanisms of
Mutagenesis, Volume 477, Number 1, 2 June 2001, pp. 97-110(14)). Poly(ADP-
ribose)
polymerase 1 (PARP1) is a key molecule in the repair of DNA single-strand
breaks (SSBs)
(de Murcia J, et al., 1997, Proc Natl Acad Sci USA 94:7303-7307; Schreiber V,
Dantzer F,
Ame JC, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang ZQ, et al.,
(1997)
Genes Dev 11:2347-2358). Knockout of SSB repair by inhibition of PARP1
function
induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality
in cancer cells
with defective homology- directed DSB repair (Bryant HE, et al., (2005) Nature
434:913-
917; Farmer H, et al., (2005) Nature 434:917-921).
[0124] BRCA1 and BRCA2 act as an integral component of the homologous
recombination machinery (HR) (Narod SA, Foulkes WD (2004) Nat Rev Cancer 4:665-
676;
Gudmundsdottir K, Ashworth A (2006) Oncogene 25:5864-5874).
[0125] Cells defective in BRCA1 or BRCA2 have a defect in the repair of double-
strand
breaks (DSB) by the mechanism of homologous recombination (HR) by gene
conversion
(Farmer H, et al., (2005) Nature 434:917-921; Narod SA, Foulkes WD (2004) Nat
Rev
Cancer 4:665-676; Gudmundsdottir K, Ashworth A (2006) Oncogene 25:5864-5874;
Helleday T, et al., (2008) Nat Rev Cancer 8:193-204). Deficiency in either of
the breast
cancer susceptibility proteins BRCA1 or BRCA2 induces profound cellular
sensitivity to the
inhibition of poly(ADP-ribose) polymerase (PARP) activity, resulting in cell
cycle arrest and
apoptosis. It has been reported that the critical role of BRCA1 and BRCA2 in
the repair of
double-strand breaks by homologous recombination (HR) is the underlying reason
for this
sensitivity, and the deficiency of RAD51, RAD54, DSS1, RPA1, NBS1, ATR, ATM,
CHK1,
CHK2, FANCD2, FANCA, or FANCC induces such sensitivity (McCabe N. et al.,
Deficiency in the repair of DNA damage by homologous recombination and
sensitivity to
poly(ADP-ribose) polymerase inhibition, Cancer research 2006, vol. 66, 8109-
8115). It has
been proposed that PARP1 inhibition can be a specific therapy for cancers with
defects in
BRCA1/2 or other HR pathway components (Helleday T, et al., (2008) Nat Rev
Cancer
8:193-204). Uterine tumors and ovarian tumors frequently harbor defects in DNA
double-
CA 02725026 2010-12-10
strand break repair through homologous recombination (HR), such as BRCA1
dysfunction
(Rottenberg S, et al., Proc Nat! Acad Sci U S A. 2008 Nov 4;105(44):17079-84).
[0126] Inhibiting the activity of a PARP molecule includes reducing the
activity of these
molecules. The term "inhibits" and its grammatical conjugations, such as
"inhibitory," is not
intended to require complete reduction in PARP activity. In some embodiments,
such
reduction is at least about 50%, at least about 75%, at least about 90%, or at
least about 95%
of the activity of the molecule in the absence of the inhibitory effect, e.g.,
in the absence of an
inhibitor, such as a nitrobenzamide compound described herein. In some
embodiments,
inhibition refers to an observable or measurable reduction in activity. In
some treatment
scenarios, the inhibition is sufficient to produce a therapeutic and/or
prophylactic benefit in
the condition being treated. The phrase "does not inhibit" and its grammatical
conjugations
does not require a complete lack of effect on the activity. For example, it
refers to situations
where there is less than about 20%, less than about 10%, and preferably less
than about 5% of
reduction in PARP activity in the presence of an inhibitor such as a
nitrobenzamide
compound of the invention.
[0127] Poly (ADP-ribose) polymerase (PARP) is an essential enzyme in DNA
repair,
thus playing a potential role in chemotherapy resistance. Targeting PARP
potentially is
thought to interrupt DNA repair, thereby enhancing taxane mediated-,
antimetabolite
mediated-, topoisomerase inhibitor-mediated, and growth factor receptor
inhibitor, e.g.,
IGF1R inhibitor-mediated, and/or platinum complex mediated-DNA replication
and/or repair
in cancer cells. PARP inhibitors may also be highly active against ovarian
cancer, uterine
cancer, and endometrial cancer with impaired function of BRCA 1 and BRCA2 or
those
patients with other DNA repair pathway defects.
[0128] 4-iodo-3-nitrobenzamide (BA) is a small molecule that acts on tumor
cells without
exerting toxic effects in normal cells. 4-iodo-3-nitrobenzamide is believed to
achieve its anti-
neoplastic effect by inhibition of PARP. 4-iodo-3-nitrobenzamide is very
lipophilic and
distributes rapidly and widely into tissues, including the brain and
cerebrospinal fluid (CSF).
It is active against a broad range of cancer cells in vitro, including against
drug resistant cell
lines. The person skilled in the art will recognize that 4-iodo-3-
nitrobenzamide may be
administered in any pharmaceutically acceptable form, e.g., as a
pharmaceutically acceptable
salt, solvate, or complex. Additionally, as 4-iodo-3-nitrobenzamide is capable
of
tautomerizing in solution, the tautomeric form of 4-iodo-3-nitrobenzamide is
intended to be
embraced by the term BA (or the equivalent 4-iodo-3-nitrobenzamide), along
with the salts,
solvates or complexes. In some embodiments, 4-iodo-3-nitrobenzamide may be
administered
41
CA 02725026 2010-12-10
in combination with a cyclodextrin, such as hydroxypropylbetacyclodextrin.
However, one
skilled in the art will recognize that other active and inactive agents may be
combined with 4-
iodo-3-nitrobenzamide; and recitation of 4-iodo-3-nitrobenzamide will, unless
otherwise
stated, include all pharmaceutically acceptable forms thereof.
[0129] Basal-like endometrial cancers have a high propensity to metastasize to
the brain;
and 4-iodo-3-nitrobenzamide is known to cross the blood-brain barrier. While
not wishing to
be bound by any particular theory, it is believed that 4-iodo-3-nitrobenzamide
achieves its
anti-neoplastic effect by inhibiting the function of PARP. In some
embodiments, 4-iodo-3-
nitrobenzamide can be used in the treatment of metastatic ovarian cancer. In
some
embodiments, 4-iodo-3-nitrobenzamide can be used in the treatment of
metastatic uterine
cancer. In some embodiments, 4-iodo-3-nitrobenzamide can be used in the
treatment of
metastatic endometrial cancer. In other embodiments, 4-iodo-3-nitrobenzamide
can be used
in the treatment of uterine, endometrial, or ovarian tumors in combination
with an anti-tumor
agent. In some embodiments, the anti-tumor agent is an antimetabolite such as
gemcitabine.
In some embodiments, the anti-tumor agent is a platinum complex such as
carboplatin. In
some embodiments, 4-iodo-3-nitrobenzamide can be used in the treatment of
uterine,
endometrial, or ovarian tumors in combination with a taxane such as
paclitaxel. In other
embodiments, 4-iodo-3-nitrobenzamide can be used in the treatment of uterine,
endometrial,
or ovarian tumors in combination with an anti-angiogenic agent. In still other
embodiments,
4-iodo-3-nitrobenzamide can be used in the treatment of uterine, endometrial,
or ovarian
tumors in combination with a topoisomerase inhibitor such as irinotecan. In
other
embodiments, 4-iodo-3-nitrobenzamide can be used in the treatment of uterine,
endometrial,
or ovarian tumors in combination with hormone therapy. In still other
embodiments, 4-iodo-
3-nitrobenzamide can be used in the treatment of uterine, endometrial, or
ovarian tumors in
combination with a growth factor receptor inhibitor including but not limited
to EGFR or
IGF1R inhibitor. In some embodiments, the uterine, endometrial, or ovarian
cancer is a
metastatic cancer.
[0130] The dosage of PARP inhibitor may vary depending upon the patient age,
height,
weight, overall health, etc. In some embodiments, the dosage of 4-iodo-3-
nitrobenzamide is
in the range of about 1 mg/kg to about 100 mg/kg, about 2 mg/kg to about 50
mg/kg, about 2
mg/kg, about 4 mg/kg, about 6 mg/kg, about 8 mg/kg, about 10 mg/kg, about 12
mg/kg,
about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35
mg/kg, about 40
mg/kg, about 50 mg/kg, about 60 mg/kg, about 75 mg/kg, about 90 mg/kg, about 1
to about
25 mg/kg, about 2 to about 70 mg/kg, about 4 to about 100 mg, about 4 to about
25 mg/kg,
42
CA 02725026 2010-12-10
about 4 to about 20 mg/kg, about 50 to about 100 mg/kg or about 25 to about 75
mg/kg. 4-
iodo-3-nitrobenzamide may be administered intravenously, e.g., by IV infusion
over about 10
to about 300 minutes, about 30 to about 180 minutes, about 45 to about 120
minutes or about
60 minutes (i.e. about 1 hour). In some embodiments, 4-iodo-3-nitrobenzamide
may
alternatively be administered orally. In this context, the term "about" has
its normal meaning
of approximately. In some embodiments, about means 10% or 5%.
[0131] The synthesis of 4-iodo-3-nitrobenzamide (4-iodo-3-nitrobenzamide) is
described
in United States Patent No. 5,464,871, which is incorporated herein by
reference in its
entirety. 4-iodo-3-nitrobenzamide may be prepared in concentrations of 10
mg/mL and may
be packaged in a convenient form, e.g., in 10 mL vials.
4-iodo-3-nitrobenzamide (BA) Metabolites
[0132] As used herein "BA" means 4-iodo-3-nitrobenzamide; "BNO" means 4-iodo-3-
nitrosobenzamide; "BNHOH" means 4-iodo-3-hydroxyaminobenzamide.
[0133] Precursor compounds useful in the present invention are of Formula (Ia)
0
11
C-NH2
RS R,
(Ia)
R4 R2
3
wherein R1, R2, R3, R4, and R5 are, independently selected from the group
consisting of
hydrogen, hydroxy, amino, nitro, iodo, (Cl -C6) alkyl, (Cl -C6) alkoxy, (C3 -
C7) cycloalkyl,
and phenyl, wherein at least two of the five R1, R2, R3, R4, and R5
substituents are always
hydrogen, at least one of the five substituents are always nitro, and at least
one substituent
positioned adjacent to a nitro is always iodo, and pharmaceutically acceptable
salts, solvates,
isomers, tautomers, metabolites, analogs, or pro-drugs thereof. R1, R2, R3,
R4, and R5 can
also be a halide such as chloro, fluoro, or bromo substituents.
[0134] A preferred precursor compound of formula la is:
43
CA 02725026 2010-12-10
O
11
C-NH2
NO2
4-iodo-3-nitrobenzamide
(BA)
[0135] Some metabolites useful in the present invention are of the Formula
(IIa):
0
11
C-NH2
RS R,
(IIa)
Ra \ R2
R3
wherein either: (1) at least one of R1, R2, R3, R4, and R5 substituent is
always a sulfur-
containing substituent, and the remaining substituents R1, R2, R3, R4, and R5
are
independently selected from the group consisting of hydrogen, hydroxy, amino,
nitro, iodo,
bromo, fluoro, chloro, (C1 -C6) alkyl, (Cl -CO alkoxy, (C3 -C7) cycloalkyl,
and phenyl,
wherein at least two of the five R1, R2, R3, R4, and R5 substituents are
always hydrogen; or (2)
at least one of R1, R2, R3, R4, and R5 substituents is not a sulfur-containing
substituent and at
least one of the five substituents R1, R2, R3, R4, and R5 is always iodo, and
wherein said iodo
is always adjacent to a R1, R2, R3, R4, or R5 group that is either a nitro, a
nitroso, a
hydroxyamino, hydroxy or an amino group; and pharmaceutically acceptable
salts, solvates,
isomers, tautomers, metabolites, analogs, or pro-drugs thereof. In some
embodiments, the
compounds of (2) are such that the iodo group is always adjacent to a R1, R2,
R3, R4 or R5
group that is a nitroso, hydroxyamino, hydroxy or amino group. In some
embodiments, the
compounds of (2) are such that the iodo group is always adjacent to a R1, R2,
R3, R4 or R5
group that is a nitroso, hydroxyamino, or amino group.
[0136] The following compositions are preferred metabolite compounds, each
represented by a chemical formula:
44
CA 02725026 2010-12-10
H2N 0
HpIV O
Il
S S N'
11
O O
HN HN
HN HN
p O
HZN HqN
O OH O OH
H O HO O
MS472 NC601
H2N 0
O
N
I I
O
R6
MS213
R6 is selected from a group consisting of hydrogen, alkyl(C1-C8), alkoxy (Cl-
C8),
isoquinolinones, indoles, thiazole, oxazole, oxadiazole, thiophene, or phenyl.
H2N 0
N"",
O
II
S
O
NH
OH
O
MS328
CA 02725026 2010-12-10
O OH
N
s 0 NH2 0 NH2 0 NH2
O
HN f/JIO
HN v OH
H2N OH NH2
NH2
HO 0 MS456 /s MS 183 MS261 /s MS 182
H2N 0 H2N 0 H2N 0
OH
OH N NH/
I I
MS263 MS276 MS278
OH
O OH O O OH
OH
=,\\\OH O
I I ~~~Z~OH
O O
HO O O HO OH
OH
OH s
O O
HN IOI HN IO
O I
HN` J~ HN`\v/ ~\ }~
\v/ ~\OH O OH
H2N MS635a H2N
MS635b
HO 0 HO O
46
CA 02725026 2010-12-10
o NH2 0 NH2
I I O1
N
NO2 NO2 0 N OH
NHZ
S 5
HO
O
HN 0 HN HO
II O
HN` OH NH
O v OH p 0 S =`d%OH
H MS471 NHZ MS414 HN H0
OH
0 pH MS692
HO O 0 OH OH
[0137] While not being limited to any one particular mechanism, the following
provides
an example for MS292 metabolism via a nitroreductase or glutathione
conjugation
mechanism:
Nitroreductase mechanism
O NH2 O NH2 O NH2
H2O
2
7~
NO2 NADPH/H+ NADP+ NO2 N
I I
NADPH/H+
NADP+
O NH2 O NH2
H2O
OH
NHZ N
NADP+ NADPH/H+ H
I I
47
CA 02725026 2010-12-10
[0138] 4-iodo-3-nitrobenzamide glutathione conjugation and metabolism:
Glutathione conjugation and metabolism LNH2 O NH2
O NH2
Glutathione Transpeptidase
NOZ --~ NOZ - ') S S
NOZ
O Glu O
Molecular Weight: 292.03 HN 0 H2N 0
BSI-201 HN` x HN` x
O \ OH ~ OH
Molecular Weight: 342.33
H2N
Gly Peptidase
HO 0
Molecular Weight: 471.44
0 NH2 NH2
N-acetyltransferase
NO 2 NO2
S HSCoA CH3COSCoA S
O
0 O
"'~N __ H H2N
OH OH
Molecular Weight: 327.31 Molecular Weight: 285.28
[0139] The present invention provides for the use of the aforesaid
nitrobenzamide
metabolite compounds for the treatment of ovarian cancer with a genetic defect
in a BRCA
gene, or a uterine cancer that is recurrent, advanced or persistent.
[0140] It has been reported that nitrobenzamide metabolite compounds have
selective
cytotoxicity upon malignant cancer cells but not upon non-malignant cancer
cells. See Rice
et at., Proc. Natl. Acad. Sci. USA 89:7703-7707 (1992), incorporated herein in
it entirety. In
one embodiment, the nitrobenzamide metabolite compounds utilized in the
methods of the
present invention may exhibit more selective toxicity towards tumor cells than
non-tumor
cells. The metabolites according to the invention may thus be administered to
a patient in
need of such treatment in conjunction with chemotherapy with at least one
taxane (e.g.,
paclitaxel or docetaxel) in addition to the at least one platinum complex
(e.g., carboplatin,
cisplatin, etc.) The dosage range for such metabolites may be in the range of
about 0.0004 to
about 0.5 mmol/kg (millimoles of metabolite per kilogram of patient body
weight), which
dosage corresponds, on a molar basis, to a range of about 0.1 to about 100
mg/kg of 4-iodo-3-
48
CA 02725026 2010-12-10
nitrobenzamide. Other effective ranges of dosages for metabolites are 0.0024-
0.5 mmol/kg
and 0.0048-0.25 mmol/kg. Such doses may be administered on a daily, every-
other-daily,
twice-weekly, weekly, bi-weekly, monthly or other suitable schedule.
Essentially the same
modes of administration may be employed for the metabolites as for 4-iodo-3-
nitrobenzamide-e.g., oral, i.v., i.p., etc.
Combination Therapy
[0141] In certain embodiments of the present invention, the methods of the
invention
further comprise treating uterine cancer, endometrial cancer, or ovarian
cancer by
administering to a subject a PARP inhibitor with or without at least one anti-
tumor agent in
combination with another anti-cancer therapy including but not limited to
surgery, radiation
therapy (e.g., X rays), gene therapy, DNA therapy, adjuvant therapy,
neoadjuvant therapy,
viral therapy, immunotherapy, RNA therapy, or nanotherapy.
[0142] Where the combination therapy further comprises a non-drug treatment,
the non-
drug treatment may be conducted at any suitable time so long as a beneficial
effect from the
co-action of the combination of the therapeutic agents and non-drug treatment
is achieved.
For example, in appropriate cases, the beneficial effect is still achieved
when the non-drug
treatment is temporally removed from the administration of the therapeutic
agents, by a
significant period of time. The conjugate and the other pharmacologically
active agent may
be administered to a patient simultaneously, sequentially or in combination.
It will be
appreciated that when using a combination of the invention, the compound of
the invention
and the other pharmacologically active agent may be in the same
pharmaceutically acceptable
carrier and therefore administered simultaneously. They may be in separate
pharmaceutical
carriers such as conventional oral dosage forms which are taken
simultaneously. The term
"combination" further refers to the case where the compounds are provided in
separate
dosage forms and are administered sequentially.
Radiation Therapy
[0143] Radiation therapy (or radiotherapy) is the medical use of ionizing
radiation as part
of cancer treatment to control malignant cells. Radiotherapy may be used for
curative or
adjuvant cancer treatment. It is used as palliative treatment (where cure is
not possible and
the aim is for local disease control or symptomatic relief) or as therapeutic
treatment (where
the therapy has survival benefit and it can be curative). Radiotherapy is used
for the treatment
of malignant tumors and may be used as the primary therapy. It is also common
to combine
radiotherapy with surgery, chemotherapy, hormone therapy or some mixture of
the three.
49
CA 02725026 2010-12-10
Most common cancer types can be treated with radiotherapy in some way. The
precise
treatment intent (curative, adjuvant, neoadjuvant, therapeutic, or palliative)
will depend on
the tumour type, location, and stage, as well as the general health of the
patient.
[0144] Radiation therapy is commonly applied to the cancerous tumor. The
radiation
fields may also include the draining lymph nodes if they are clinically or
radiologically
involved with tumor, or if there is thought to be a risk of subclinical
malignant spread. It is
necessary to include a margin of normal tissue around the tumor to allow for
uncertainties in
daily set-up and internal tumor motion.
[0145] Radiation therapy works by damaging the DNA of cells. The damage is
caused by
a photon, electron, proton, neutron, or ion beam directly or indirectly
ionizing the atoms
which make up the DNA chain. Indirect ionization happens as a result of the
ionization of
water, forming free radicals, notably hydroxyl radicals, which then damage the
DNA. In the
most common forms of radiation therapy, most of the radiation effect is
through free radicals.
Because cells have mechanisms for repairing DNA damage, breaking the DNA on
both
strands proves to be the most significant technique in modifying cell
characteristics. Because
cancer cells generally are undifferentiated and stem cell-like, they reproduce
more, and have
a diminished ability to repair sub-lethal damage compared to most healthy
differentiated
cells. The DNA damage is inherited through cell division, accumulating damage
to the cancer
cells, causing them to die or reproduce more slowly. Proton radiotherapy works
by sending
protons with varying kinetic energy to precisely stop at the tumor.
[0146] Gamma rays are also used to treat some types of cancer including
uterine,
endometrial, and ovarian cancers. In the procedure called gamma-knife surgery,
multiple
concentrated beams of gamma rays are directed on the growth in order to kill
the cancerous
cells. The beams are aimed from different angles to focus the radiation on the
growth while
minimizing damage to the surrounding tissues.
Gene Therapy Agents
[0147] Gene therapy agents insert copies of genes into a specific set of a
patient's cells,
and can target both cancer and non-cancer cells. The goal of gene therapy can
be to replace
altered genes with functional genes, to stimulate a patient's immune response
to cancer, to
make cancer cells more sensitive to chemotherapy, to place "suicide" genes
into cancer cells,
or to inhibit angiogenesis. Genes may be delivered to target cells using
viruses, liposomes, or
other carriers or vectors. This may be done by injecting the gene-carrier
composition into the
CA 02725026 2010-12-10
patient directly, or ex vivo, with infected cells being introduced back into a
patient. Such
compositions are suitable for use in the present invention.
Adiuvant therapy
[0148] Adjuvant therapy is a treatment given after the primary treatment to
increase the
chances of a cure. Adjuvant therapy may include chemotherapy, radiation
therapy, hormone
therapy, or biological therapy.
[0149] Adjuvant chemotherapy is effective for patients with advanced uterine
cancer or
ovarian cancer. The combination of doxorubicin and cisplatin achieves overall
response rates
ranging from 34 to 60%, and the addition of paclitaxel seems to improve the
outcome of
patients with advanced disease, but it induces a significantly higher
toxicity. A Gynecologic
Oncology Study Group phase-III study is currently exploring the triplet
paclitaxel+doxorubicin+cisplatin plus G-CSF vs. the less toxic combination of
paclitaxel+carboplatin. Ongoing and planned phase-III trials are evaluating
newer
combination chemotherapy regimens, a combination of irradiation and
chemotherapy and the
implementation of targeted therapies with the goal of improving the tumor
control rate and
quality of life.
[0150] Adjuvant radiation therapy (RT) - Adjuvant radiation therapy
significantly
reduces the risk that the uterine cancer will recur locally (i.e., in the
pelvis or vagina). In
general, there are two ways of delivering RT: it may be given as vaginal
brachytherapy or as
external beam RT (EBRT). In vaginal brachytherapy, brachytherapy delivers RT
directly to
the vaginal tissues from a source that is temporarily placed inside the body.
This allows high
doses of radiation to be delivered to the area where cancer cells are most
likely to be found.
With external beam radiation therapy (EBRT), the source of the radiation is
outside the body.
[0151] Various therapies including but not limited to hormone therapy, e.g.,
tamoxifen, or
gonadotropin-releasing hormone (GnRH) analogues, and radioactive monoclonal
antibody
therapy have been used to treat ovarian cancer.
Neoadjuvant therapy
[0152] Neoadjuvant therapy refers to a treatment given before the primary
treatment.
Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and
hormone
therapy. Neoadjuvant chemotherapy in gynecological cancers is an approach that
is shown to
have positive effects on survival. It increases the rate of resectability in
ovarian and cervical
cancers and thus contributes to survival (Ayhan A. et. al. European journal of
gynaecological
oncology. 2006, vol. 27).
51
CA 02725026 2010-12-10
Oncolytic viral therapy
[0153] Viral therapy for cancer utilizes a type of viruses called oncolytic
viruses. An
oncolytic virus is a virus that is able to infect and lyse cancer cells, while
leaving normal cells
unharmed, making them potentially useful in cancer therapy. Replication of
oncolytic viruses
both facilitates tumor cell destruction and also produces dose amplification
at the tumor site.
They may also act as vectors for anticancer genes, allowing them to be
specifically delivered
to the tumor site.
[0154] There are two main approaches for generating tumor selectivity:
transductional
and non-transductional targeting. Transductional targeting involves modifying
the specificity
of viral coat protein, thus increasing entry into target cells while reducing
entry to non-target
cells. Non-transductional targeting involves altering the genome of the virus
so it can only
replicate in cancer cells. This can be done by either transcription targeting,
where genes
essential for viral replication are placed under the control of a tumor-
specific promoter, or by
attenuation, which involves introducing deletions into the viral genome that
eliminate
functions that are dispensable in cancer cells, but not in normal cells. There
are also other,
slightly more obscure methods.
[0155] Chen et al (2001) used CV706, a prostate-specific adenovirus, in
conjunction with
radiotherapy on prostate cancer in mice. The combined treatment results in a
synergistic
increase in cell death, as well as a significant increase in viral burst size
(the number of virus
particles released from each cell lysis).
[0156] ONYX-015 has undergone trials in conjunction with chemotherapy. The
combined treatment gives a greater response than either treatment alone, but
the results have
not been entirely conclusive. ONYX-015 has shown promise in conjunction with
radiotherapy.
[0157] Viral agents administered intravenously can be particularly effective
against
metastatic cancers, which are especially difficult to treat conventionally.
However,
bloodborne viruses can be deactivated by antibodies and cleared from the blood
stream
quickly e.g., by Kupffer cells (extremely active phagocytic cells in the
liver, which are
responsible for adenovirus clearance). Avoidance of the immune system until
the tumour is
destroyed could be the biggest obstacle to the success of oncolytic virus
therapy. To date, no
technique used to evade the immune system is entirely satisfactory. It is in
conjunction with
conventional cancer therapies that oncolytic viruses show the most promise,
since combined
therapies operate synergistically with no apparent negative effects.
52
CA 02725026 2010-12-10
[0158] The specificity and flexibility of oncolytic viruses means they have
the potential
to treat a wide range of cancers including uterine cancer, endometrial cancer,
and ovarian
cancer with minimal side effects. Oncolytic viruses have the potential to
solve the problem of
selectively killing cancer cells.
Nanotherapy
[0159] Nanometer-sized particles have novel optical, electronic, and
structural properties
that are not available from either individual molecules or bulk solids. When
linked with
tumor-targeting moieties, such as tumor-specific ligands or monoclonal
antibodies, these
nanoparticles can be used to target cancer-specific receptors, tumor antigens
(biomarkers),
and tumor vasculatures with high affinity and precision. The formulation and
manufacturing
process for cancer nanotherapy is disclosed in patent US7179484, and article
M. N. Khalid,
P. Simard, D. Hoarau, A. Dragomir, J. Leroux, Long Circulating Poly(Ethylene
Glycol)Decorated Lipid Nanocapsules Deliver Docetaxel to Solid Tumors,
Pharmaceutical
Research, 23(4), 2006, all of which are herein incorporated by reference in
their entireties.
RNA therapy
[0160] RNA including but not limited to siRNA, shRNA, microRNA may be used to
modulate gene expression and treat cancers. Double stranded oligonucleotides
are formed by
the assembly of two distinct oligonucleotide sequences where the
oligonucleotide sequence
of one strand is complementary to the oligonucleotide sequence of the second
strand; such
double stranded oligonucleotides are generally assembled from two separate
oligonucleotides
(e.g., siRNA), or from a single molecule that folds on itself to form a double
stranded
structure (e.g., shRNA or short hairpin RNA). These double stranded
oligonucleotides known
in the art all have a common feature in that each strand of the duplex has a
distinct nucleotide
sequence, wherein only one nucleotide sequence region (guide sequence or the
antisense
sequence) has complementarity to a target nucleic acid sequence and the other
strand (sense
sequence) comprises nucleotide sequence that is homologous to the target
nucleic acid
sequence.
[0161] MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23
nucleotides in length, which regulate gene expression. miRNAs are encoded by
genes that are
transcribed from DNA but not translated into protein (non-coding RNA); instead
they are
processed from primary transcripts known as pri-miRNA to short stem-loop
structures called
pre-miRNA and finally to functional miRNA. Mature miRNA molecules are
partially
53
CA 02725026 2010-12-10
complementary to one or more messenger RNA (mRNA) molecules, and their main
function
is to downregulate gene expression.
[0162] Certain RNA inhibiting agents may be utilized to inhibit the expression
or
translation of messenger RNA ("mRNA") that is associated with a cancer
phenotype.
Examples of such agents suitable for use herein include, but are not limited
to, short
interfering RNA ("siRNA"), ribozymes, and antisense oligonucleotides. Specific
examples
of RNA inhibiting agents suitable for use herein include, but are not limited
to, Cand5, Sirna-
027, fomivirsen, and angiozyme.
Small Molecule Enzymatic Inhibitors
[0163] Certain small molecule therapeutic agents are able to target the
tyrosine kinase
enzymatic activity or downstream signal transduction signals of certain cell
receptors such as
epidermal growth factor receptor ("EGFR") or vascular endothelial growth
factor receptor
("VEGFR"). Such targeting by small molecule therapeutics can result in anti-
cancer effects.
Examples of such agents suitable for use herein include, but are not limited
to, imatinib,
gefitinib, erlotinib, lapatinib, canertinib, ZD6474, sorafenib (BAY 43-9006),
ERB-569, and
their analogues and derivatives.
Anti-Metastatic Agents
[0164] The process whereby cancer cells spread from the site of the original
tumor to
other locations around the body is termed cancer metastasis. Certain agents
have anti-
metastatic properties, designed to inhibit the spread of cancer cells.
Examples of such agents
suitable for use herein include, but are not limited to, marimastat,
bevacizumab, trastuzumab,
rituximab, erlotinib, MMI-166, GRN163L, hunter-killer peptides, tissue
inhibitors of
metalloproteinases (TIMPs), their analogues, derivatives and variants.
Chemopreventative agents
[0165] Certain pharmaceutical agents can be used to prevent initial
occurrences of cancer,
or to prevent recurrence or metastasis. Administration with such
chemopreventative agents
in combination with eflornithine-NSAID conjugates of the invention can act to
both treat and
prevent the recurrence of cancer. Examples of chemopreventative agents
suitable for use
herein include, but are not limited to, tamoxifen, raloxifene, tibolone,
bisphosphonate,
ibandronate, estrogen receptor modulators, aromatase inhibitors (letrozole,
anastrozole),
luteinizing hormone-releasing hormone agonists, goserelin, vitamin A, retinal,
retinoic acid,
fenretinide, 9-cis-retinoid acid, 13-cis-retinoid acid, all-trans-retinoic
acid, isotretinoin,
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CA 02725026 2010-12-10
tretinoid, vitamin B6, vitamin B 12, vitamin C, vitamin D, vitamin E,
cyclooxygenase
inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), aspirin,
ibuprofen, celecoxib,
polyphenols, polyphenol E, green tea extract, folic acid, glucaric acid,
interferon-alpha,
anethole dithiolethione, zinc, pyridoxine, finasteride, doxazosin, selenium,
indole-3-carbinal,
alpha-difluoromethylornithine, carotenoids, beta-carotene, lycopene,
antioxidants, coenzyme
Q10, flavonoids, quercetin, curcumin, catechins, epigallocatechin gallate, N-
acetylcysteine,
indole-3-carbinol, inositol hexaphosphate, isoflavones, glucanic acid,
rosemary, soy, saw
palmetto, and calcium. An additional example of chemopreventative agents
suitable for use
in the present invention is cancer vaccines. These can be created through
immunizing a
patient with all or part of a cancer cell type that is targeted by the
vaccination process.
Clinical Efficacy
[0166] Clinical efficacy may be measured by any method known in the art. In
some
embodiments, clinical efficacy of the therapeutic treatments described herein
may be
determined by measuring the clinical benefit rate (CBR). The clinical benefit
rate is
measured by determining the sum of the percentage of patients who are in
complete
remission (CR), the number of patients who are in partial remission (PR) and
the number of
patients having stable disease (SD) at a time point at least 6 months out from
the end of
therapy. The shorthand for this formula is CBR = CR + PR + SD > 6 months. The
CBR for
combination therapy with paclitaxel and carboplatin is 45%. Thus, the CBR for
triple
combination therapy with a taxane, platinum complex and PARP inhibitor (e.g.,
paclitaxel,
carboplatin and 4-iodo-3-nitrobenzamide; CBRGCB) may be compared to that of
the double
combination therapy with paclitaxel and carboplatin (CBRGC). Similarly, the
CBR for
combination therapy with an antimetabolite (e.g., gemcitabine), a platinum
compound (e.g.,
carboplatin), and a PARP inhibitor (e.g., 4-iodo-3-nitrobenzamide)
(CBRGEM/CARBO/BA) may
be compared to that of the double combination therapy with an antimetabolite
(e.g.,
gemcitabine) and a platinum compound (e.g., carboplatin) (CBRGEM/CARBO)= In
some
embodiments, CBRGEM/cARBO/BA is at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%,
80% or more. In some embodiments, the CBR is at least about 30%, at least
about 40%, or at
least about 50%.
[0167] In some embodiments disclosed herein, the methods include pre-
determining that
a cancer is treatable by PARP inhibitors. Some such methods comprise
identifying a level of
PARP in a uterine, endometrial, or ovarian cancer sample of a patient,
determining whether
the level of PARP expression in the sample is greater than a pre-determined
value, and, if the
CA 02725026 2010-12-10
PARP expression is greater than said predetermined value, treating the patient
with a
combination of an anti-tumor agent described herein and a PARP inhibitor such
as 4-iodo-3-
nitrobenzamide. In other embodiments, the methods comprise identifying a level
of PARP
in a uterine, endometrial, or ovarian cancer sample of a patient, determining
whether the level
of PARP expression in the sample is greater than a pre-determined value, and,
if the PARP
expression is greater than said predetermined value, treating the patient with
a PARP
inhibitor, such as 4-iodo-3-nitrobenzamide.
[0168] Uterine tumors in women who inherit faults in either the BRCA1 or BRCA2
genes occur because the tumor cells have lost a specific mechanism that repair
damaged
DNA. BRCA1 and BRCA2 are important for DNA double-strand break repair by
homologous recombination, and mutations in these genes predispose to uterine
and other
cancers. PARP is involved in base excision repair, a pathway in the repair of
DNA single-
strand breaks. BRCA1 or BRCA2 dysfunction sensitizes cells to the inhibition
of PARP
enzymatic activity, resulting in chromosomal instability, cell cycle arrest
and subsequent
apoptosis (Jones C, Plummer ER. PARP inhibitors and cancer therapy - early
results and
potential applications. Br J Radiol. 2008 Oct;81 Spec No 1:S2-5; Drew Y,
Calvert H. The
potential of PARP inhibitors in genetic breast and ovarian cancers. Ann N Y
Acad Sci. 2008
Sep;1138:136-45; Farmer H, et al., Targeting the DNA repair defect in BRCA
mutant cells
as a therapeutic strategy. Nature. 2005 Apr 14;434(7035):917-21).
[0169] Patients deficient in BRCA genes may have up-regulated levels of PARP.
PARP
up-regulation may be an indicator of other defective DNA-repair pathways and
unrecognized
BRCA-like genetic defects. Assessment of PARP gene expression and impaired DNA
repair
especially defective homologous recombination DNA repair can be used as an
indicator of
tumor sensitivity to PARP inhibitor. Hence, in some embodiments, treatment of
uterine
cancer can be enhanced not only by determining the HR and/or HER2 status of
the cancer,
but also by identifying early onset of cancer in BRCA and homologous
recombination DNA
repair deficient patients by measuring the level of PARP. The BRCA and
homologous
recombination DNA repair deficient patients treatable by PARP inhibitors can
be identified if
PARP is up-regulated. Further, such homologous recombination DNA repair
deficient
patients can be treated with PARP inhibitors.
[0170] In some embodiments, a sample is collected from a patient having a
uterine lesion
suspected of being cancerous. While such sample may be any available
biological tissue, in
most cases the sample will be a portion of the suspected uterine lesion,
whether obtained by
laparoscopy or open surgery (e.g., a hysterectomy). PARP expression may then
be analyzed
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CA 02725026 2010-12-10
and, if the PARP expression is above a predetermined level (e.g., is up-
regulated compared to
normal tissue) the patient may be treated with a PARP inhibitor in combination
with an
antimetabolite (e.g., gemcitabine), a platinum compound (e.g., carboplatin),
and a PARP
inhibitor (e.g., 4-iodo-3-nitrobenzamide). It is thus to be understood that,
while embodiments
described herein are directed to treatment of endometrial cancer, recurrent,
advanced, or
persistent uterine cancer, and ovarian cancer in association with a BRCA-
defect, in some
embodiments, the uterine or ovarian cancer need not have these characteristics
so long as the
threshold PARP up-regulation is satisfied.
[0171] In some embodiments, tumors that are homologous recombination deficient
are
identified by evaluating levels of PARP expression. If up-regulation of PARP
is observed,
such tumors can be treated with PARP inhibitors. Another embodiment is a
method for
treating a homologous recombination deficient cancer comprising evaluating
level of PARP
expression and, if overexpression is observed, the cancer is treated with a
PARP inhibitor.
Sample collection, preparation and separation
[0172] Biological samples may be collected from a variety of sources from a
patient
including a body fluid sample, or a tissue sample. Samples collected can be
human normal
and tumor samples, nipple aspirants. The samples can be collected from
individuals
repeatedly over a longitudinal period of time (e.g., about once a day, once a
week, once a
month, biannually or annually). Obtaining numerous samples from an individual
over a
period of time can be used to verify results from earlier detections and/or to
identify an
alteration in biological pattern as a result of, for example, disease
progression, drug
treatment, etc.
[0173] Sample preparation and separation can involve any of the procedures,
depending
on the type of sample collected and/or analysis of PARP. Such procedures
include, by way
of example only, concentration, dilution, adjustment of pH, removal of high
abundance
polypeptides (e.g., albumin, gamma globulin, and transferin, etc.), addition
of preservatives
and calibrants, addition of protease inhibitors, addition of denaturants,
desalting of samples,
concentration of sample proteins, extraction and purification of lipids.
[0174] The sample preparation can also isolate molecules that are bound in non-
covalent
complexes to other protein (e.g., carrier proteins). This process may isolate
those molecules
bound to a specific carrier protein (e.g., albumin), or use a more general
process, such as the
release of bound molecules from all carrier proteins via protein denaturation,
for example
using an acid, followed by removal of the carrier proteins.
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CA 02725026 2010-12-10
[0175] Removal of undesired proteins (e.g., high abundance, uninformative, or
undetectable proteins) from a sample can be achieved using high affinity
reagents, high
molecular weight filters, ultracentrifugation and/or electrodialysis. High
affinity reagents
include antibodies or other reagents (e.g., aptamers) that selectively bind to
high abundance
proteins. Sample preparation could also include ion exchange chromatography,
metal ion
affinity chromatography, gel filtration, hydrophobic chromatography,
chromatofocusing,
adsorption chromatography, isoelectric focusing and related techniques.
Molecular weight
filters include membranes that separate molecules on the basis of size and
molecular weight.
Such filters may further employ reverse osmosis, nanofiltration,
ultrafiltration and
microfiltration.
[0176] Ultracentrifugation is a method for removing undesired polypeptides
from a
sample. Ultracentrifugation is the centrifugation of a sample at about 15,000-
60,000 rpm
while monitoring with an optical system the sedimentation (or lack thereof) of
particles.
Electrodialysis is a procedure which uses an electromembrane or semipermable
membrane in
a process in which ions are transported through semi-permeable membranes from
one
solution to another under the influence of a potential gradient. Since the
membranes used in
electrodialysis may have the ability to selectively transport ions having
positive or negative
charge, reject ions of the opposite charge, or to allow species to migrate
through a
semipermable membrane based on size and charge, it renders electrodialysis
useful for
concentration, removal, or separation of electrolytes.
[0177] Separation and purification in the present invention may include any
procedure
known in the art, such as capillary electrophoresis (e.g., in capillary or on-
chip) or
chromatography (e.g., in capillary, column or on a chip). Electrophoresis is a
method which
can be used to separate ionic molecules under the influence of an electric
field.
Electrophoresis can be conducted in a gel, capillary, or in a microchannel on
a chip.
Examples of gels used for electrophoresis include starch, acrylamide,
polyethylene oxides,
agarose, or combinations thereof. A gel can be modified by its cross-linking,
addition of
detergents, or denaturants, immobilization of enzymes or antibodies (affinity
electrophoresis)
or substrates (zymography) and incorporation of a pH gradient. Examples of
capillaries used
for electrophoresis include capillaries that interface with an electrospray.
[0178] Capillary electrophoresis (CE) is preferred for separating complex
hydrophilic
molecules and highly charged solutes. CE technology can also be implemented on
microfluidic chips. Depending on the types of capillary and buffers used, CE
can be further
segmented into separation techniques such as capillary zone electrophoresis
(CZE), capillary
58
CA 02725026 2010-12-10
isoelectric focusing (CLEF), capillary isotachophoresis (cITP) and capillary
electrochromatography (CEC). An embodiment to couple CE techniques to
electrospray
ionization involves the use of volatile solutions, for example, aqueous
mixtures containing a
volatile acid and/or base and an organic such as an alcohol or acetonitrile.
[0179] Capillary isotachophoresis (cITP) is a technique in which the analytes
move
through the capillary at a constant speed but are nevertheless separated by
their respective
mobilities. Capillary zone electrophoresis (CZE), also known as free-solution
CE (FSCE), is
based on differences in the electrophoretic mobility of the species,
determined by the charge
on the molecule, and the frictional resistance the molecule encounters during
migration which
is often directly proportional to the size of the molecule. Capillary
isoelectric focusing
(CLEF) allows weakly-ionizable amphoteric molecules, to be separated by
electrophoresis in
a pH gradient. CEC is a hybrid technique between traditional high performance
liquid
chromatography (HPLC) and CE.
[0180] Separation and purification techniques used in the present invention
include any
chromatography procedures known in the art. Chromatography can be based on the
differential adsorption and elution of certain analytes or partitioning of
analytes between
mobile and stationary phases. Different examples of chromatography include,
but not limited
to, liquid chromatography (LC), gas chromatography (GC), high performance
liquid
chromatography (HPLC) etc.
Measuring levels of PARP expression
[0181] The poly (ADP-ribose) polymerase (PARP) is also known as poly (ADP-
ribose)
synthase and poly ADP-ribosyltransferase. PARP catalyzes the formation of poly
(ADP-
ribose) polymers which can attach to cellular proteins (as well as to itself)
and thereby
modify the activities of those proteins. The enzyme plays a role in enhancing
DNA repair,
but it also plays a role in regulation of transcription, cell proliferation,
and chromatin
remodeling (for review see: D. D'amours et al., "Poly (ADP-ribosylation
reactions in the
regulation of nuclear functions," Biochem. J. 342: 249-268 (1999)).
[0182] PARP-1 comprises an N-terminal DNA binding domain, an automodification
domain and a C-terminal catalytic domain and various cellular proteins
interact with PARP-1.
The N-terminal DNA binding domain contains two zinc finger motifs..
Transcription
enhancer factor-1 (TEF-1), retinoid X receptor a, DNA polymerase a, X-ray
repair cross-
complementing factor-1 (XRCC1) and PARP-1 itself interact with PARP-1 in this
domain.
The automodification domain contains a BRCT motif, one of the protein-protein
interaction
59
CA 02725026 2010-12-10
modules. This motif is originally found in the C-terminus of BRCA1 (uterine
cancer
susceptibility protein 1) and is present in various proteins related to DNA
repair,
recombination and cell-cycle checkpoint control. POU-homeodomain-containing
octamer
transcription factor-1 (Oct-1), Yin Yang (YY)1 and ubiquitin-conjugating
enzyme 9 (ubc9)
could interact with this BRCT motif in PARP-1.
[0183] More than 15 members of the PARP family of genes are present in the
mammalian genome. PARP family proteins and poly(ADP-ribose) glycohydrolase
(PARG),
which degrades poly(ADP-ribose) to ADP-ribose, could be involved in a variety
of cell
regulatory functions including DNA damage response and transcriptional
regulation and may
be related to carcinogenesis and the biology of cancer in many respects.
[0184] Several PARP family proteins have been identified. Tankyrase has been
found as
an interacting protein of telomere regulatory factor 1 (TRF-1) and is involved
in telomere
regulation. Vault PARP (VPARP) is a component in the vault complex, which acts
as a
nuclear-cytoplasmic transporter. PARP-2, PARP-3 and 2,3,7,8-tetrachlorodibenzo-
p-dioxin
inducible PARP (TiPARP) have also been identified. Therefore, poly (ADP-
ribose)
metabolism could be related to a variety of cell regulatory functions.
[0185] A member of this gene family is PARP-1. The PARP-1 gene product is
expressed
at high levels in the nuclei of cells and is dependent upon DNA damage for
activation.
Without being bound by any theory, it is believed that PARP-1 binds to DNA
single or
double stranded breaks through an amino terminal DNA binding domain. The
binding
activates the carboxy terminal catalytic domain and results in the formation
of polymers of
ADP-ribose on target molecules. PARP-1 is itself a target of poly ADP-
ribosylation by
virtue of a centrally located automodification domain. The ribosylation of
PARP-1 causes
dissociation of the PARP-1 molecules from the DNA. The entire process of
binding,
ribosylation, and dissociation occurs very rapidly. It has been suggested that
this transient
binding of PARP-1 to sites of DNA damage results in the recruitment of DNA
repair
machinery or may act to suppress the recombination long enough for the
recruitment of repair
machinery.
[0186] The source of ADP-ribose for the PARP reaction is nicotinamide
adenosine
dinucleotide (NAD). NAD is synthesized in cells from cellular ATP stores and
thus high
levels of activation of PARP activity can rapidly lead to depletion of
cellular energy stores. It
has been demonstrated that induction of PARP activity can lead to cell death
that is correlated
with depletion of cellular NAD and ATP pools. PARP activity is induced in many
instances
of oxidative stress or during inflammation. For example, during reperfusion of
ischemic
CA 02725026 2010-12-10
tissues reactive nitric oxide is generated and nitric oxide results in the
generation of
additional reactive oxygen species including hydrogen peroxide, peroxynitrate
and hydroxyl
radical. These latter species can directly damage DNA and the resulting damage
induces
activation of PARP activity. Frequently, it appears that sufficient activation
of PARP activity
occurs such that the cellular energy stores are depleted and the cell dies. A
similar
mechanism is believed to operate during inflammation when endothelial cells
and pro-
inflammatory cells synthesize nitric oxide which results in oxidative DNA
damage in
surrounding cells and the subsequent activation of PARP activity. The cell
death that results
from PARP activation is believed to be a major contributing factor in the
extent of tissue
damage that results from ischemia-reperfusion injury or from inflammation.
[0187] In some embodiments, the level of PARP in a sample from a patient is
compared
to predetermined standard sample. The sample from the patient is typically
from a diseased
tissue, such as cancer cells or tissues. The standard sample can be from the
same patient or
from a different subject. The standard sample is typically a normal, non-
diseased sample.
However, in some embodiments, such as for staging of disease or for evaluating
the efficacy
of treatment, the standard sample is from a diseased tissue. The standard
sample can be a
combination of samples from several different subjects. In some embodiments,
the level of
PARP from a patient is compared to a pre-determined level. This pre-determined
level is
typically obtained from normal samples. As described herein, a "pre-determined
PARP
level" may be a level of PARP used to, by way of example only, evaluate a
patient that may
be selected for treatment, evaluate a response to a PARP inhibitor treatment,
evaluate a
response to a combination of a PARP inhibitor and a second therapeutic agent
treatment,
and/or diagnose a patient for cancer, inflammation, pain and/or related
conditions. A pre-
determined PARP level may be determined in populations of patients with or
without cancer.
The pre-determined PARP level can be a single number, equally applicable to
every patient,
or the pre-determined PARP level can vary according to specific subpopulations
of patients.
For example, men might have a different pre-determined PARP level than women;
non-
smokers may have a different pre-determined PARP level than smokers. Age,
weight, and
height of a patient may affect the pre-determined PARP level of the
individual. Furthermore,
the pre-determined PARP level can be a level determined for each patient
individually. The
pre-determined PARP level can be any suitable standard. For example, the pre-
determined
PARP level can be obtained from the same or a different human for whom a
patient selection
is being assessed. In one embodiment, the pre-determined PARP level can be
obtained from
a previous assessment of the same patient. In such a manner, the progress of
the selection of
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CA 02725026 2010-12-10
the patient can be monitored over time. In addition, the standard can be
obtained from an
assessment of another human or multiple humans, e.g., selected groups of
humans. In such a
manner, the extent of the selection of the human for whom selection is being
assessed can be
compared to suitable other humans, e.g., other humans who are in a similar
situation to the
human of interest, such as those suffering from similar or the same
condition(s).
[0188] In some embodiments of the present invention the change of PARP from
the pre-
determined level is about 0.5 fold, about 1.0 fold, about 1.5 fold, about 2.0
fold, about 2.5
fold, about 3.0 fold, about 3.5 fold, about 4.0 fold, about 4.5 fold, or about
5.0 fold. In some
embodiments is fold change is less than about 1, less than about 5, less than
about 10, less
than about 20, less than about 30, less than about 40, or less than about 50.
In other
embodiments, the changes in PARP level compared to a predetermined level is
more than
about 1, more than about 5, more than about 10, more than about 20, more than
about 30,
more than about 40, or more than about 50. Preferred fold changes from a pre-
determined
level are about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, and about
3Ø
[0189] The analysis of PARP levels in patients is particularly valuable and
informative,
as it allows the physician to more effectively select the best treatments, as
well as to utilize
more aggressive treatments and therapy regimens based on the up-regulated or
down-
regulated level of PARP. More aggressive treatment, or combination treatments
and
regimens, can serve to counteract poor patient prognosis and overall survival
time. Armed
with this information, the medical practitioner can choose to provide certain
types of
treatment such as treatment with PARP inhibitors, and/or more aggressive
therapy.
[0190] In monitoring a patient's PARP levels, over a period of time, which may
be days,
weeks, months, and in some cases, years, or various intervals thereof, the
patient's body fluid
sample, e.g., serum or plasma, can be collected at intervals, as determined by
the practitioner,
such as a physician or clinician, to determine the levels of PARP, and
compared to the levels
in normal individuals over the course or treatment or disease. For example,
patient samples
can be taken and monitored every month, every two months, or combinations of
one, two, or
three month intervals according to the invention. In addition, the PARP levels
of the patient
obtained over time can be conveniently compared with each other, as well as
with the PARP
values, of normal controls, during the monitoring period, thereby providing
the patient's own
PARP values, as an internal, or personal, control for long-term PARP
monitoring.
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CA 02725026 2010-12-10
Techniques for Analysis of PARP
[0191] Previous studies have shown increased PARP activity in ovarian cancers,
hepatocellular carcinomas, and rectal tumors, compared with normal healthy
control tissues,
as well as in human peripheral blood lymphocytes from leukemia patients
(Yalcintepe L, et
al., J Med Biol Res 2005;38:361-5. Singh N. et al., Cancer Lett 1991;58:131-5;
Nomura F, et
al., J Gastroenterol Hepatol 2000;15:529-35). Thus, assessing PARP activity in
a tumor cell
would allow one of ordinary skill in the art to determine whether a particular
tumor would
benefit from treatment with a PARP inhibitor such as 4-iodo-3-nitrobenzamide.
See, e.g.,
U.S. Patent Publication No. US 2009/0123419 Al, which is incorporated herein
by reference.
[0192] The analysis of the PARP may include analysis of PARP gene expression,
including an analysis of DNA, RNA, analysis of the level of PARP and/or
analysis of the
activity of PARP including a level of mono- and poly-ADP-ribozylation. Without
limiting
the scope of the present invention, any number of techniques known in the art
can be
employed for the analysis of PARP and they are all within the scope of the
present invention.
Some of the examples of such detection technique are given below but these
examples are in
no way limiting to the various detection techniques that can be used in the
present invention.
[0193] Gene Expression Profiling: Methods of gene expression profiling include
methods based on hybridization analysis of polynucleotides,
polyribonucleotides methods
based on sequencing of polynucleotides, polyribonucleotides and proteomics-
based methods.
The most commonly used methods known in the art for the quantification of mRNA
expression in a sample include northern blotting and in situ hybridization
(Parker & Barnes,
Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays
(Hod,
Biotechniques 13:852-854 (1992)); and PCR-based methods, such as reverse
transcription
polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264
(1992)).
Alternatively, antibodies may be employed that can recognize specific
duplexes, including
DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein
duplexes.
Representative methods for sequencing-based gene expression analysis include
Serial
Analysis of Gene Expression (SAGE), and gene expression analysis by massively
parallel
signature sequencing (MPSS), Comparative Genome Hybridisation (CGH), Chromatin
Immunoprecipitation (ChIP), Single nucleotide polymorphism (SNP) and SNP
arrays,
Fluorescent in situ Hybridization (FISH), Protein binding arrays and DNA
microarray (also
commonly known as gene or genome chip, DNA chip, or gene array),
RNAmicroarrays.
[0194] Reverse Transcriptase PCR (RT-PCR): One of the most sensitive and most
flexible quantitative PCR-based gene expression profiling methods is RT-PCR,
which can be
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CA 02725026 2010-12-10
used to compare mRNA levels in different sample populations, in normal and
tumor tissues,
with or without drug treatment, to characterize patterns of gene expression,
to discriminate
between closely related mRNAs, and to analyze RNA structure.
The first step is the isolation of mRNA from a target sample. For example, the
starting
material can be typically total RNA isolated from human tumors or tumor cell
lines, and
corresponding normal tissues or cell lines, respectively. Thus RNA can be
isolated from a
variety of normal and diseased cells and tissues, for example tumors,
including breast, lung,
colorectal, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis,
ovary, uterus, etc.,
or tumor cell lines,. If the source of mRNA is a primary tumor, mRNA can be
extracted, for
example, from frozen or archived fixed tissues, for example paraffin-embedded
and fixed
(e.g., formalin-fixed) tissue samples. General methods for mRNA extraction are
well known
in the art and are disclosed in standard textbooks of molecular biology,
including Ausubel et
al., Current Protocols of Molecular Biology, John Wiley and Sons (1997).
[0195] In particular, RNA isolation can be performed using purification kit,
buffer set and
protease from commercial manufacturers, according to the manufacturer's
instructions. RNA
prepared from tumor can be isolated, for example, by cesium chloride density
gradient
centrifugation. As RNA cannot serve as a template for PCR, the first step in
gene expression
profiling by RT-PCR is the reverse transcription of the RNA template into
cDNA, followed
by its exponential amplification in a PCR reaction. The two most commonly used
reverse
transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT)
and Moloney
murine leukemia virus reverse transcriptase (MMLV-RT). The reverse
transcription step is
typically primed using specific primers, random hexamers, or oligo-dT primers,
depending on
the circumstances and the goal of expression profiling. The derived cDNA can
then be used
as a template in the subsequent PCR reaction.
[0196] To minimize errors and the effect of sample-to-sample variation, RT-PCR
is
usually performed using an internal standard. The ideal internal standard is
expressed at a
constant level among different tissues, and is unaffected by the experimental
treatment.
RNAs most frequently used to normalize patterns of gene expression are mRNAs
for the
housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and (3-
actin.
[0197] A more recent variation of the RT-PCR technique is the real time
quantitative
PCR, which measures PCR product accumulation through a dual-labeled
fluorigenic probe.
Real time PCR is compatible both with quantitative competitive PCR, where
internal
competitor for each target sequence is used for normalization, and with
quantitative
64
CA 02725026 2010-12-10
comparative PCR using a normalization gene contained within the sample, or a
housekeeping
gene for RT-PCR.
[0198] Fluorescence Microscopy: Some embodiments of the invention include
fluorescence microscopy for analysis of PARP. Fluorescence microscopy enables
the
molecular composition of the structures being observed to be identified
through the use of
fluorescently-labeled probes of high chemical specificity such as antibodies.
It can be done
by directly conjugating a fluorophore to a protein and introducing this back
into a cell.
Fluorescent analogue may behave like the native protein and can therefore
serve to reveal the
distribution and behavior of this protein in the cell. Along with NMR,
infrared spectroscopy,
circular dichroism and other techniques, protein intrinsic fluorescence decay
and its
associated observation of fluorescence anisotropy, collisional quenching and
resonance
energy transfer are techniques for protein detection. The naturally
fluorescent proteins can be
used as fluorescent probes. The jellyfish aequorea victoria produces a
naturally fluorescent
protein known as green fluorescent protein (GFP). The fusion of these
fluorescent probes to
a target protein enables visualization by fluorescence microscopy and
quantification by flow
cytometry.
By way of example only, some of the probes are labels such as, fluorescein and
its
derivatives, carboxyfluoresceins, rhodamines and their derivatives, atto
labels, fluorescent red
and fluorescent orange: cy3/cy5 alternatives, lanthanide complexes with long
lifetimes, long
wavelength labels - up to 800 nm, DY cyanine labels, and phycobili proteins.
By way of
example only, some of the probes are conjugates such as, isothiocyanate
conjugates,
streptavidin conjugates, and biotin conjugates. By way of example only, some
of the probes
are enzyme substrates such as, fluorogenic and chromogenic substrates. By way
of example
only, some of the probes are fluorochromes such as, FITC (green fluorescence,
excitation/emission = 506/529 nm), rhodamine B (orange fluorescence,
excitation/emission =
560/584 nm), and nile blue A (red fluorescence, excitation/emission = 636/686
nm).
Fluorescent nanoparticles can be used for various types of immunoassays.
Fluorescent
nanoparticles are based on different materials, such as, polyacrylonitrile,
and polystyrene etc.
Fluorescent molecular rotors are sensors of microenvironmental restriction
that become
fluorescent when their rotation is constrained. Few examples of molecular
constraint include
increased dye (aggregation), binding to antibodies, or being trapped in the
polymerization of
actin. IEF (isoelectric focusing) is an analytical tool for the separation of
ampholytes, mainly
proteins. An advantage for IEF-gel electrophoresis with fluorescent IEF-marker
is the
CA 02725026 2010-12-10
possibility to directly observe the formation of gradient. Fluorescent IEF-
marker can also be
detected by UV-absorption at 280 nm (20 C).
[0199] A peptide library can be synthesized on solid supports and, by using
coloring
receptors, subsequent dyed solid supports can be selected one by one. If
receptors cannot
indicate any color, their binding antibodies can be dyed. The method can not
only be used on
protein receptors, but also on screening binding ligands of synthesized
artificial receptors and
screening new metal binding ligands as well. Automated methods for HTS and
FACS
(fluorescence activated cell sorter) can also be used. A FACS machine
originally runs cells
through a capillary tube and separates cells by detecting their fluorescent
intensities.
[0200] Immunoassays: Some embodiments of the invention include immunoassay for
the analysis of PARP. In immunoblotting like the western blot of
electrophoretically
separated proteins a single protein can be identified by its antibody.
Immunoassay can be
competitive binding immunoassay where analyte competes with a labeled antigen
for a
limited pool of antibody molecules (e.g., radioimmunoassay, EMIT). Immunoassay
can be
non-competitive where antibody is present in excess and is labeled. As analyte
antigen
complex is increased, the amount of labeled antibody-antigen complex may also
increase
(e.g., ELISA). Antibodies can be polyclonal if produced by antigen injection
into an
experimental animal, or monoclonal if produced by cell fusion and cell culture
techniques. In
immunoassay, the antibody may serve as a specific reagent for the analyte
antigen.
[0201] Without limiting the scope and content of the present invention, some
of the types
of immunoassays are, by way of example only, RIAs (radioimmunoassay), enzyme
immunoassays like ELISA (enzyme-linked immunosorbent assay), EMIT (enzyme
multiplied
immunoassay technique), microparticle enzyme immunoassay (MEIA), LIA
(luminescent
immunoassay), and FIA (fluorescent immunoassay). These techniques can be used
to detect
biological substances in the nasal specimen. The antibodies - either used as
primary or
secondary ones - can be labeled with radioisotopes (e.g., 1251), fluorescent
dyes (e.g., FITC)
or enzymes (e.g., HRP or AP) which may catalyse fluorogenic or luminogenic
reactions.
[0202] Biotin, or vitamin H is a co-enzyme which inherits a specific affinity
towards
avidin and streptavidin. This interaction makes biotinylated peptides a useful
tool in various
biotechnology assays for quality and quantity testing. To improve
biotin/streptavidin
recognition by minimizing steric hindrances, it can be necessary to enlarge
the distance
between biotin and the peptide itself. This can be achieved by coupling a
spacer molecule
(e.g., 6-nitrohexanoic acid) between biotin and the peptide.
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CA 02725026 2010-12-10
[0203] The biotin quantitation assay for biotinylated proteins provides a
sensitive
fluorometric assay for accurately determining the number of biotin labels on a
protein.
Biotinylated peptides are widely used in a variety of biomedical screening
systems requiring
immobilization of at least one of the interaction partners onto streptavidin
coated beads,
membranes, glass slides or microtiter plates. The assay is based on the
displacement of a
ligand tagged with a quencher dye from the biotin binding sites of a reagent.
To expose any
biotin groups in a multiply labeled protein that are sterically restricted and
inaccessible to the
reagent, the protein can be treated with protease for digesting the protein.
[0204] EMIT is a competitive binding immunoassay that avoids the usual
separation step.
A type of immunoassay in which the protein is labeled with an enzyme, and the
enzyme-
protein-antibody complex is enzymatically inactive, allowing quantitation of
unlabelled
protein. Some embodiments of the invention include ELISA to analyze PARR ELISA
is
based on selective antibodies attached to solid supports combined with enzyme
reactions to
produce systems capable of detecting low levels of proteins. It is also known
as enzyme
immunoassay or EIA. The protein is detected by antibodies that have been made
against it,
that is, for which it is the antigen. Monoclonal antibodies are often used.
[0205] The test may require the antibodies to be fixed to a solid surface,
such as the inner
surface of a test tube, and a preparation of the same antibodies coupled to an
enzyme. The
enzyme may be one (e.g., (3-galactosidase) that produces a colored product
from a colorless
substrate. The test, for example, may be performed by filling the tube with
the antigen
solution (e.g., protein) to be assayed. Any antigen molecule present may bind
to the
immobilized antibody molecules. The antibody-enzyme conjugate may be added to
the
reaction mixture. The antibody part of the conjugate binds to any antigen
molecules that are
bound previously, creating an antibody-antigen-antibody "sandwich". After
washing away
any unbound conjugate, the substrate solution may be added. After a set
interval, the reaction
is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product
formed is
measured in a spectrophotometer. The intensity of color is proportional to the
concentration
of bound antigen.
[0206] ELISA can also be adapted to measure the concentration of antibodies,
in which
case, the wells are coated with the appropriate antigen. The solution (e.g.,
serum) containing
antibody may be added. After it has had time to bind to the immobilized
antigen, an enzyme-
conjugated anti-immunoglobulin may be added, consisting of an antibody against
the
antibodies being tested for. After washing away unreacted reagent, the
substrate may be
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CA 02725026 2010-12-10
added. The intensity of the color produced is proportional to the amount of
enzyme-labeled
antibodies bound (and thus to the concentration of the antibodies being
assayed).
[0207] Some embodiments of the invention include radioimmunoassays to analyze
PARP. Radioactive isotopes can be used to study in vivo metabolism,
distribution, and
binding of small amount of compounds. Radioactive isotopes of 'H, 12C,3 I p,
32S, and 127I in
body are used such as 3H 14C 32P, 35S, and 1251. In receptor fixation method
in 96 well
plates, receptors may be fixed in each well by using antibody or chemical
methods and
radioactive labeled ligands may be added to each well to induce binding.
Unbound ligands
may be washed out and then the standard can be determined by quantitative
analysis of
radioactivity of bound ligands or that of washed-out ligands. Then, addition
of screening
target compounds may induce competitive binding reaction with receptors. If
the compounds
show higher affinity to receptors than standard radioactive ligands, most of
radioactive
ligands would not bind to receptors and may be left in solution. Therefore, by
analyzing
quantity of bound radioactive ligands (or washed-out ligands), testing
compounds' affinity to
receptors can be indicated.
[0208] The filter membrane method may be needed when receptors cannot be fixed
to 96
well plates or when ligand binding needs to be done in solution phase. In
other words, after
ligand-receptor binding reaction in solution, if the reaction solution is
filtered through
nitrocellulose filter paper, small molecules including ligands may go through
it and only
protein receptors may be left on the paper. Only ligands that strongly bound
to receptors may
stay on the filter paper and the relative affinity of added compounds can be
identified by
quantitative analysis of the standard radioactive ligands.
[0209] Some embodiments of the invention include fluorescence immunoassays for
the
analysis of PARP. Fluorescence based immunological methods are based upon the
competitive binding of labeled ligands versus unlabeled ones on highly
specific receptor
sites. The fluorescence technique can be used for immunoassays based on
changes in
fluorescence lifetime with changing analyte concentration. This technique may
work with
short lifetime dyes like fluorescein isothiocyanate (FITC) (the donor) whose
fluorescence
may be quenched by energy transfer to eosin (the acceptor). A number of
photoluminescent
compounds may be used, such as cyanines, oxazines, thiazines, porphyrins,
phthalocyanines,
fluorescent infrared-emitting polynuclear aromatic hydrocarbons,
phycobiliproteins,
squaraines and organo-metallic complexes, hydrocarbons and azo dyes.
[0210] Fluorescence based immunological methods can be, for example,
heterogenous or
homogenous. Heterogenous immunoassays comprise physical separation of bound
from free
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CA 02725026 2010-12-10
labeled analyte. The analyte or antibody may be attached to a solid surface.
The technique
can be competitive (for a higher selectivity) or noncompetitive (for a higher
sensitivity).
Detection can be direct (only one type of antibody used) or indirect (a second
type of
antibody is used). Homogenous immunoassays comprise no physical separation.
Double-
antibody fluorophore-labeled antigen participates in an equilibrium reaction
with antibodies
directed against both the antigen and the fluorophore. Labeled and unlabeled
antigen may
compete for a limited number of anti-antigen antibodies.
[0211] Some of the fluorescence immunoassay methods include simple
fluorescence
labeling method, fluorescence resonance energy transfer (FRET), time resolved
fluorescence
(TRF), and scanning probe microscopy (SPM). The simple fluorescence labeling
method can
be used for receptor-ligand binding, enzymatic activity by using pertinent
fluorescence, and
as a fluorescent indicator of various in vivo physiological changes such as
pH, ion
concentration, and electric pressure. TRF is a method that selectively
measures fluorescence
of the lanthanide series after the emission of other fluorescent molecules is
finished. TRF can
be used with FRET and the lanthanide series can become donors or acceptors. In
scanning
probe microscopy, in the capture phase, for example, at least one monoclonal
antibody is
adhered to a solid phase and a scanning probe microscope is utilized to detect
antigen/antibody complexes which may be present on the surface of the solid
phase. The use
of scanning tunneling microscopy eliminates the need for labels which normally
is utilized in
many immunoassay systems to detect antigen/antibody complexes.
[0212] Protein identification methods: By way of example only, protein
identification
methods include low-throughput sequencing through Edman degradation, mass
spectrometry
techniques, peptide mass fingerprinting, de novo sequencing, and antibody-
based assays. The
protein quantification assays include fluorescent dye gel staining, tagging or
chemical
modification methods (i.e. isotope-coded affinity tags (ICATS), combined
fractional diagonal
chromatography (COFRADIC)). The purified protein may also be used for
determination of
three-dimensional crystal structure, which can be used for modeling
intermolecular
interactions. Common methods for determining three-dimensional crystal
structure include
x-ray crystallography and NMR spectroscopy. Characteristics indicative of the
three-
dimensional structure of proteins can be probed with mass spectrometry. By
using chemical
crosslinking to couple parts of the protein that are close in space, but far
apart in sequence,
information about the overall structure can be inferred. By following the
exchange of amide
protons with deuterium from the solvent, it is possible to probe the solvent
accessibility of
various parts of the protein.
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[0213] In one embodiment, fluorescence-activated cell-sorting (FACS) is used
to identify
PARP expressing cells. FACS is a specialised type of flow cytometry. It
provides a method
for sorting a heterogenous mixture of biological cells into two or more
containers, one cell at
a time, based upon the specific light scattering and fluorescent
characteristics of each cell. It
provides quantitative recording of fluorescent signals from individual cells
as well as physical
separation of cells of particular interest. In yet another embodiment,
microfluidic based
devices are used to evaluate PARP expression.
[0214] Mass spectrometry can also be used to characterize PARP from patient
samples.
The two methods for ionization of whole proteins are electrospray ionization
(ESI) and
matrix-assisted laser desorption/ionization (MALDI). In the first, intact
proteins are ionized
by either of the two techniques described above, and then introduced to a mass
analyser. In
the second, proteins are enzymatically digested into smaller peptides using an
agent such as
trypsin or pepsin. Other proteolytic digest agents are also used. The
collection of peptide
products are then introduced to the mass analyser. This is often referred to
as the "bottom-
up" approach of protein analysis.
[0215] Whole protein mass analysis is conducted using either time-of-flight
(TOF) MS,
or Fourier transform ion cyclotron resonance (FT-ICR). The instrument used for
peptide
mass analysis is the quadrupole ion trap. Multiple stage quadrupole-time-of-
flight and
MALDI time-of-flight instruments also find use in this application.
[0216] Two methods used to fractionate proteins, or their peptide products
from an
enzymatic digestion. The first method fractionates whole proteins and is
called two-
dimensional gel electrophoresis. The second method, high performance liquid
chromatography is used to fractionate peptides after enzymatic digestion. In
some situations,
it may be necessary to combine both of these techniques.
[0217] There are two ways mass spectroscopy can be used to identify proteins.
Peptide
mass uses the masses of proteolytic peptides as input to a search of a
database of predicted
masses that would arise from digestion of a list of known proteins. If a
protein sequence in
the reference list gives rise to a significant number of predicted masses that
match the
experimental values, there is some evidence that this protein is present in
the original sample.
[0218] Tandem MS is also a method for identifying proteins. Collision-induced
dissociation is used in mainstream applications to generate a set of fragments
from a specific
peptide ion. The fragmentation process primarily gives rise to cleavage
products that break
along peptide bonds.
CA 02725026 2010-12-10
[0219] A number of different algorithmic approaches have been described to
identify
peptides and proteins from tandem mass spectrometry (MS/MS), peptide de novo
sequencing
and sequence tag based searching. One option that combines a comprehensive
range of data
analysis features is PEAKS. Other existing mass spec analysis software
include: Peptide
fragment fingerprinting SEQUEST, Mascot, OMSSA and X!Tandem).
[0220] Proteins can also be quantified by mass spectrometry. Typically, stable
(e.g., non-
radioactive) heavier isotopes of carbon (C13) or nitrogen (N15) are
incorporated into one
sample while the other one is labelled with corresponding light isotopes
(e.g., C12 and N14).
The two samples are mixed before the analysis. Peptides derived from the
different samples
can be distinguished due to their mass difference. The ratio of their peak
intensities
corresponds to the relative abundance ratio of the peptides (and proteins).
The methods for
isotope labelling are SILAC (stable isotope labelling with amino acids in cell
culture),
trypsin-catalyzed 018 labeling, ICAT (isotope coded affinity tagging), ITRAQ
(isotope tags
for relative and absolute quantitation). "Semi-quantitative" mass spectrometry
can be
performed without labeling of samples. Typically, this is done with MALDI
analysis (in
linear mode). The peak intensity, or the peak area, from individual molecules
(typically
proteins) is here correlated to the amount of protein in the sample. However,
the individual
signal depends on the primary structure of the protein, on the complexity of
the sample, and
on the settings of the instrument.
[0221] N-terminal sequencing aids in the identification of unknown proteins,
confirm
recombinant protein identity and fidelity (reading frame, translation start
point, etc.), aid the
interpretation of NMR and crystallographic data, demonstrate degrees of
identity between
proteins, or provide data for the design of synthetic peptides for antibody
generation, etc. N-
terminal sequencing utilises the Edman degradative chemistry, sequentially
removing amino
acid residues from the N-terminus of the protein and identifying them by
reverse-phase
HPLC. Sensitivity can be at the level of 100s femtomoles and long sequence
reads (20-40
residues) can often be obtained from a few 10s of picomoles of starting
material. Pure
proteins (>90%) can generate easily interpreted data, but insufficiently
purified protein
mixtures may also provide useful data, subject to rigorous data
interpretation. N-terminally
modified (especially acetylated) proteins cannot be sequenced directly, as the
absence of a
free primary amino-group prevents the Edman chemistry. However, limited
proteolysis of
the blocked protein (e.g., using cyanogen bromide) may allow a mixture of
amino acids to be
generated in each cycle of the instrument, which can be subjected to database
analysis in
order to interpret meaningful sequence information. C-terminal sequencing is a
post-
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CA 02725026 2010-12-10
translational modification, affecting the structure and activity of a protein.
Various disease
situations can be associated with impaired protein processing and C-terminal
sequencing
provides an additional tool for the investigation of protein structure and
processing
mechanisms.
Formulations, Routes of Administration, and Dosing Regimen
[0222] In some embodiments are provided formulations (e.g., pharmaceutical
formulations) comprising 4-iodo-3-nitrobenzamide, a metabolite thereof, or a
pharmaceutically acceptable salt or solvate thereof, an antimetabolite (e.g.,
gemcitabine) and
a platinum compound (e.g., carboplatin) and a carrier, such as a
pharmaceutically acceptable
carrier. The formulations may include optical isomers, diastereomers,
carriers, or
pharmaceutically acceptable salts of the compounds disclosed herein. In some
embodiments,
the carrier is a cyclodextrin, or a derivative thereof, e.g., hydroxypropyl- -
B-cyclodextrin
(HPBCD). In some embodiments the formulations are formulated for intravenous
administration.
[0223] The pharmaceutical compositions of the present invention may be
provided as a
prodrug and/or may be allowed to interconvert to 4-iodo-3-nitrobenzamide form
in vivo after
administration. That is, either 4-iodo-3-nitrobenzamide or metabolites thereof
or
pharmaceutically acceptable salts may be used in developing a formulation for
use in the
present invention. 4-iodo-3-nitrobenzamide (or a metabolite thereof), an
antimetabolite (e.g.,
gemcitabine) and a platinum compound (e.g., carboplatin) provided herein may
be formulated
in separate formulations or in the same formulation. 4-iodo-3-nitrobenzamide
(or a
metabolite thereof), an antimetabolite (e.g., gemcitabine) and a platinum
compound (e.g.,
carboplatin) provided herein may be administered through different
administration route or
using same administration routes.
[0224] A formulation may comprise both the 4-iodo-3-nitrobenzamide compound
and
acid forms in particular proportions, depending on the relative potencies of
each and the
intended indication. The two forms may be formulated together or in different
formulations.
They may be in the same dosage unit e.g. in one cream, suppository, tablet,
capsule, or packet
of powder to be dissolved in a beverage; or each form may be formulated in a
separate unit,
e.g., two creams, two suppositories, two tablets, two capsules, a tablet and a
liquid for
dissolving the tablet, a packet of powder and a liquid for dissolving the
powder, etc.
[0225] 4-iodo-3-nitrobenzamide (or a metabolite thereof), an antimetabolite
(e.g.,
gemcitabine) and a platinum compound (e.g., carboplatin) provided herein may
be co-
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CA 02725026 2010-12-10
administered to the patient. Co-administration is meant to include
simultaneous or sequential
administration of the compounds individually or in combination (more than one
compound),
such as described herein. 4-iodo-3-nitrobenzamide (or a metabolite thereof),
an
antimetabolite (e.g., gemcitabine) and/or a platinum compound (e.g.,
carboplatin) provided
herein may be continuously or not continuously given to a patient. "Not
continuously" means
that the compound or composition provided herein is not administered to the
patient over a
period of time, e.g., there is a resting period when the patient does not
receive the compound
or composition. It may be that one compound is administered continuously
administered to a
patient while the second compound is not administered continuously
administered to the
patient.
[0226] The pharmaceutical compositions of the 4-iodo-3-nitrobenzamide, an
antimetabolite (e.g., gemcitabine) and a platinum compound (e.g., carboplatin)
can be
combined with other active ingredients, such as other chemotherapeutic agents
as described
herein. The tthree compounds and/or forms of a compound may be formulated
together, in
the same dosage unit e.g., in one cream, suppository, tablet, capsule, or
packet of powder to
be dissolved in a beverage; or each form may be formulated in separate units,
e.g., three
creams, three suppositories, three tablets, three capsules, a tablet and a
liquid for dissolving
the tablet, a packet of powder and a liquid for dissolving the powder, etc.
[0227] The term "pharmaceutically acceptable salt" means those salts which
retain the
biological effectiveness and properties of the compounds used in the present
invention, and
which are not biologically or otherwise undesirable. For example, a
pharmaceutically
acceptable salt does not interfere with the beneficial effect of the compound
of the invention
in treating platinum-sensitive ovarian cancer (e.g., recurrent ovarian
cancer).
[0228] Typical salts are those of the inorganic ions, such as, for example,
sodium,
potassium, calcium and magnesium ions. Such salts include salts with inorganic
or organic
acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric
acid, sulfuric acid,
methanesulfonic acid, p toluenesulfonic acid, acetic acid, fumaric acid,
succinic acid, lactic
acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid. In
addition, where
compounds contain a carboxy group or other acidic group, it may be converted
into a
pharmaceutically acceptable addition salt with inorganic or organic bases.
Examples of
suitable bases include sodium hydroxide, potassium hydroxide, ammonia,
cyclohexylamine,
dicyclohexyl-amine, ethanolamine, diethanolamine and triethanolamine.
[0229] For injection, the 4-iodo-3-nitrobenzamide or pharmaceutically
acceptable salt
thereof may be formulated for administration in aqueous solutions, preferably
in
73
CA 02725026 2010-12-10
physiologically compatible buffers such as phosphate buffers, Hank's solution,
or Ringer's
solution. Such compositions may also include one or more excipients, for
example,
preservatives, solubilizers, fillers, lubricants, stabilizers, albumin, and
the like. Formulations
of 4-iodo-3-nitrobenzamide are described in US Pat. Publ. No. 2008/0176946 Al,
which is
incorporated by reference in its entirety, particularly with reference to
intravenous (e.g.,
hydroxypropyl-(3-cyclodextrin, etc.) and oral (e.g., sodium lauryl sulfate,
etc.) formulations.
In some embodiments, the 4-iodo-3-nitrobenzamide is formulated in 25% (w/v)
hydroxypropyl-
0-cyclodextrin and 10 mM phosphate buffer for intravenous administration as
described in U.S.
Patent Application No. 12/510,969, filed July 28, 2009, which is incorporated
herein by
reference.
[0230] Additional methods of formulation, such as for the antimetabolite
(e.g.,
gemcitabine) and the platinum compound (e.g., carboplatin) described herein,
are known in
the art, for example, as disclosed in Remington's Pharmaceutical Sciences,
latest edition,
Mack Publishing Co., Easton, PA. Compositions described herein may also be
formulated
for transmucosal administration, buccal administration, for administration by
inhalation, for
parental administration, for transdermal administration, and rectal
administration.
[0231] Pharmaceutical compositions suitable for use as described herein
include
compositions wherein the active ingredients are present in an effective
amount, i.e., in an
amount effective to achieve therapeutic and/or prophylactic benefit in at
least one of the
platinum-sensitive ovarian cancers (e.g., recurrent ovarian cancer) described
herein. The
actual amount effective for a particular administration will depend on the
platinum-sensitive
ovarian cancer (e.g., recurrent ovarian cancer) being treated, the condition
of the subject, the
formulation, and the route of administration, as well as other factors known
to those of skill
in the art in view of the specific teaching provided herein. In light of the
disclosure herein,
optimization of an effective amount of 4-iodo-3-nitrobenzamide, antimetabolite
(e.g.,
gemcitabine), and/or platinum compound (e.g., carboplatin) provided herein,
within the
ranges specified, may be determined.
[0232] In some embodiments, the composition is administered in unit dosage
form. In
some embodiments, the unit dosage form is adapted for oral or parenteral
administration. In
some embodiments, upon administration of the composition, at least one
therapeutic effect is
obtained, said at least one therapeutic effect being reduction in size of a
tumor, reduction in
metastasis, complete remission, partial remission, pathologic complete
response, increase in
overall response rate,or stable disease. In some embodiments, upon
administration of the
composition, an improvement of clinical benefit rate (CBR = CR + PR + SD > 6
months) is
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CA 02725026 2010-12-10
obtained as compared to treatment with the antimetabolite (e.g., gemcitabine)
and the
platinum compound (e.g., carboplatin) but without 4-iodo-3-nitrobenzamide or
the metabolite
thereof or the pharmaceutically acceptable salt thereof. In some embodiments,
the
improvement of clinical benefit rate is at least about 20%. In some
embodiments, the
improvement of clinical benefit rate is at least about 25%, 30%, 35%, 40%,
45%, 50%, 55%,
60%, 65%, 70%, 75% or more.
[0233] The compositions described herein may be administered to a patient
through
appropriate route, such as, but are not limited to intradermal, intramuscular,
intraperitoneal,
intravenous, intraarterial, subcutaneous, intranasal, epidural, and oral
routes. In some
embodiments, the composition or compound(s) provided herein is administered by
the
parenteral route, e.g., intravenously, intraperitoneally, subcutaneously,
intradermally, or
intramuscularly.
[0234] Compositions may be administered by any convenient route, for example
by
infusion or bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be administered in
combination with
other biologically active agents, e.g., such as described herein.
Administration can be
systemic or local. In addition, it may be desirable to introduce the
pharmaceutical
compositions of the invention into the central nervous system by any suitable
route, including
intraventricular and intrathecal injection; intraventricular injection may be
facilitated by an
intraventricular catheter, for example, attached to a reservoir, such as an
Ommaya reservoir.
[0235] The dosage of 4-iodo-3-nitrobenzamide ("BA") or a metabolite thereof or
a
pharmaceutically acceptable salt thereof may vary depending upon the patient
age, height,
weight, overall health, etc. In some embodiments, the dosage of 4-iodo-3-
nitrobenzamide is
in the range of about 1 mg/kg to about 100 mg/kg, about 2 mg/kg to about 50
mg/kg, about 2
to 10 mg/kg, about 4 to 8 mg/kg, about 5 to 7 mg/kg, about 1 to about 25
mg/kg, about 2 to
about 70 mg/kg, about 4 to about 100 mg/kg, about 4 to about 25 mg/kg, about 4
to about 20
mg/kg, about 50 to about 100 mg/kg or about 25 to about 75 mg/kg. In some
embodiments,
the dosage of 4-iodo-3-nitrobenzamide is about any of 1 mg/kg, 2 mg/kg, 3
mg/kg, 4 mg/kg,
mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 30
mg/kg, 50
mg/kg, 75 mg/kg, or 100 mg/kg. 4-iodo-3-nitrobenzamide or its metabolite may
be
administered intravenously, e.g., by IV infusion over about 10 to about 300
minutes, about 30
to about 180 minutes, about 45 to about 120 minutes or about 60 minutes (i.e.
about 1 hour).
In some embodiments, 4-iodo-3-nitrobenzamide may alternatively be administered
orally.
CA 02725026 2010-12-10
= [0236] The dosage of an antimetabolite provided herein (e.g., gemcitabine)
may vary
depending upon the patient age, height, weight, overall health, etc. In some
embodiments, the
dosage of the antimetabolite (e.g., gemcitabine) provided herein is in the
range of about 10
mg/m2 to about 1000 mg/m2, about 25mg/m2 to about 500 mg/m2, about 50 mg/m2 to
about
200 mg/m2, about 75 mg/m2 to about 200 mg/m2. In some embodiments, the dosage
of the
antimetabolite (e.g., gemcitabine) provided herein is about any of 50 mg/m2,
75 mg/m2, 100
mg/m2, 125 mg/m2, 150 mg/m2, 175 mg/m2, 200 mg/m2, 250 mg/m2, or 300 mg/m2. An
antimetabolite (e.g., gemcitabine) provided herein may be administered
intravenously, e.g.,
by IV infusion over about 10 to about 500 minutes, about 10 to about 300
minutes, about 30
to about 180 minutes, about 45 to about 120 minutes or about 60 minutes (i.e.
about 1 hour).
In some embodiments, an alkylating agent provided herein may alternatively be
administered
orally.
[0237] The dosage of a platinum compound provided herein (e.g., carboplatin)
may vary
depending upon the patient age, height, weight, overall health, etc. The
dosage of a platinum
compound, e.g., carboplatin, is determined by calculating the area under the
blood plasma
concentration versus time curve (AUC) in mg/mL=minute by methods known to
those skilled
in the cancer chemotherapy art, taking into account the patient's renal
activity estimated by
measuring creatinine clearance or glomerular filtration rate. In some
embodiments, the
dosage of carboplatin for combination treatment along with an antimetabolite
(e.g.,
gemcitabine) and a PARP inhibitor (e.g., 4-iodo-3-nitrobenzamide) is
calculated to provide
an AUC of about 0.1-6 mg/ml-min, about 1-3 mg/ml-min, about 1.5 to about 2.5
mg/ml-min,
about 1.75 to about 2.25 mg/ml-min or about 2 mg/ml-min. (AUC 2, for example,
is
shorthand for 2 mg/ml-minute). Alternatively, the dosage of platinum compound
(e.g.,
carboplatin) is calculated based on the patient's body surface area. In some
embodiments, a
suitable dose of platinum compound (e.g., carboplatin) is about 10 to about
400 mg/m2, e.g.,
about 360 mg/m2. Platinum complexes platinum compound (e.g., carboplatin) are
normally
administered intravenously (IV) over a period of about about 10 to about 500
minutes, about
to about 300 minutes, about 30 to about 180 minutes, about 45 to about 120
minutes or
about 60 minutes. In this context, the term "about" has its normal meaning of
approximately.
In some embodiments, about means 10% or 5%.
[0238] In some cases, a beneficial effect is achieved when the administration
of the
antimetabolite (e.g., gemcitabine) and the platinum compound (e.g.,
carboplatin) is
temporally removed from the administration of the 4-iodo-3-nitrobenazmide (or
pharmaceutically acceptable salt or solvate thereof, or metabolite thereof) by
a significant
76
CA 02725026 2010-12-10
period of time (e.g., about 12 hours, about 24 hours, about 36 hours, about 48
hours, etc.), or,
for example, when administration is spaced apart by at least 1 day, 2 days, 3
days, 4 days, 5
days, 6 days, 7 days, 8 days, 9 days, 10 days, etc.). For example,
administration may be on
different days of a treatment cycle, such as the treatment cycles described
herein. The
interval between administration of the 4-iodo-3-nitrobenzamide, the
antimetabolite (e.g.,
gemcitabine), and the platinum compound (e.g., carboplatin) may vary within a
treatment
cycle (e.g., administration is not always spaced apart by 1 day, but may be at
intervals of 1
day followed by an interval of 3 days, etc.). Similarly, at certain times
during the treatment
cycle, the 4-iodo-3-nitrobenzamide, the antimetabolite (e.g., gemcitabine),
and the platinum
compound (e.g., carboplatin) may be administered at the same time, and at
other points
during the treatment administered at different times.
[0239] In some embodiments, the treatment includes 1 cycle, 2 cycles, 3
cycles, 4 cycles,
cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles or 10 cycles. As used here, the
term "cycle"
means "treatment cycle." In some embodiments, the treatment includes at most
any of 2
cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles,
or 10 cycles.
[0240] In some embodiments, the treatment comprises a treatment cycle of at
least about
any of 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks,
8 weeks, 9
weeks, 10 weeks, 12 weeks, or 15 weeks.
[0241] 4-iodo-3-nitrobenzamide may be administered every day of the treatment
cycle, or
administered on certain days but not on every day of the treatment cycle. In
some
embodiments, 4-iodo-3-nitrobenzamide is administered daily, once a week, twice
a week,
three times a week, four times a week, five times a week, six times a week,
once 10 days,
once two weeks, once three weeks, once four weeks, once six weeks, or once
eight weeks. 4-
iodo-3-nitrobenzamide may be administered on the selected days of each
treatment cycle, for
example, 4-iodo-3-nitrobenzamide is administered daily for the period of 3 (or
4, 5, 6, 7, 8, 9,
10) days of the treatment cycle, and 4-iodo-3-nitrobenzamide is not
administered on other
days of the treatment cycle.
[0242] An antimetabolite (e.g., gemcitabine) provided herein may be
administered daily,
e.g., every day of the treatment cycle, or administered on certain days but
not on every day of
the treatment cycle. In some embodiments, the antimetabolite (e.g.,
gemcitabine) provided
herein is administered daily, once a week, twice a week, three times a week,
four times a
week, five times a week, six times a week, once every 10 days, once every two
weeks, once
every three weeks, once every four weeks, once every six weeks, or once every
eight weeks.
An antimetabolite (e.g., gemcitabine) provided herein may be administered on
the selected
77
CA 02725026 2010-12-10
days of each treatment cycle, for example, the antimetabolite (e.g.,
gemcitabine) is
administered daily for the period of 3 (or 4, 5, 6, 7, 8, 9, 10) days of the
treatment cycle, and
the antimetabolite (e.g., gemcitabine) is not administered on other days of
the treatment
cycle.
[0243] A platinum compound (e.g., carboplatin) provided herein may be
administered
daily, e.g., every day of the treatment cycle, or administered on certain days
but not on every
day of the treatment cycle. In some embodiments, the platinum compound (e.g.,
carboplatin)
provided herein is administered daily, once a week, twice a week, three times
a week, four
times a week, five times a week, six times a week, once every 10 days, once
every two
weeks, once every three weeks, once every four weeks, once every six weeks, or
once every
eight weeks. A platinum compound (e.g., carboplatin) provided herein may be
administered
on the selected days of each treatment cycle, for example, the platinum
compound (e.g.,
carboplatin) is administered daily for the period of 3 (or 4, 5, 6, 7, 8, 9,
10) days of the
treatment cycle, and the platinum compound (e.g., carboplatin) is not
administered on other
days of the treatment cycle.
[0244] In some embodiments of any one of the methods for treating platinum-
sensitive
recurrent ovarian cancer provided herein, the method comprises 6 or fewer
dosing cycles,
wherein each cycle contains a period of 21 days. In some embodiments, 4-iodo-3-
nitrobenzamide or the pharmaceutically acceptable salt thereof is administered
at about 5.1
mg/kg to about 8.6 mg/kg on days 1, 4, 8, and 11 of each cycle, the
antimetabolite (e.g.,
gemcitabine is administered at 1000 mg/m2 daily on days 1 and 8 of each cycle,
and the
platinum compound (e.g., carboplatin) is administered at 4 mg/ml-minute (AUC
4) on day 1
of each cycle.
Kits
[0245] Also provided are kits for administration of 4-iodo-3-nitrobenzamide or
a
metabolite thereof or a pharmaceutically acceptable salt thereof, gemcitabine
and carboplatin
as provided herein.
[0246] In certain embodiments the kits may include a dosage amount of at least
one
composition as disclosed herein. Kits may further comprise suitable packaging
and/or
instructions for use of the formulation. Kits may also comprise a means for
the delivery of
the formulation thereof.
[0247] The kits may include other pharmaceutical agents (such as the side-
effect limiting
agents, chemotherapy agents, gene therapy agents, DNA therapy agents, RNA
therapy agents,
78
CA 02725026 2010-12-10
viral therapy agents, nanotherapy agents, small molecule enzymatic inhibitors,
anti-metastatic
agents, etc.), for use in conjunction with 4-iodo-3-nitrobenzamide or a
metabolite thereof or a
pharmaceutically acceptable salt thereof, an antimetabolite (e.g.,
gemcitabine) provided
herein, and a platinum compound (e.g., carboplatin) provided herein. These
agents may be
provided in a separate form, or mixed with 4-iodo-3-nitrobenzamide or a
metabolite thereof
or a pharmaceutically acceptable salt thereof, an antimetabolite (e.g.,
gemcitabine) provided
herein, and a platinum compound (e.g., carboplatin) provided herein, provided
such mixing
does not reduce the effectiveness of 4-iodo-3-nitrobenzamide (or a metabolite
thereof or a
pharmaceutically acceptable salt thereof), an antimetabolite (e.g.,
gemcitabine) provided
herein or a platinum compound (e.g., carboplatin) provided herein, and is
compatible with the
route of administration. Similarly, the kits may include additional agents for
adjunctive
therapy or other agents known to the skilled artisan as effective in the
treatment or prevention
of platinum-sensitive ovarian cancer (e.g., recurrent ovarian cancer)
described herein.
[0248] The kits may optionally include appropriate instructions for
preparation and
administration of the composition, side effects of the composition, and any
other relevant
information. The instructions may be in any suitable format, including, but
not limited to,
printed matter, videotape, computer readable disk, optical disc or directions
to internet-based
instructions.
[0249] In another aspect, provided are kits for treating a patient who suffers
from or is
susceptible to the platinum-sensitive ovarian cancer (e.g., recurrent ovarian
cancer) described
herein, comprising a first container comprising a dosage amount of a
formulation as disclosed
herein, and instructions for use. The container may be any of those known in
the art and
appropriate for storage and delivery of intravenous formulation. In certain
embodiments the
kit further comprises a second container comprising a pharmaceutically
acceptable carrier,
diluent, adjuvant, etc. for preparation of the composition to be administered
to the patient.
[0250] Kits may also be provided that contain sufficient dosages of the
inhibitor
(including formulation thereof) as disclosed herein to provide effective
treatment for a patient
for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks,
4 weeks, 6
weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9
months or
more.
[0251] Kits may also include multiple doses of the compounds and instructions
for use
and packaged in quantities sufficient for storage and use in pharmacies, for
example, hospital
pharmacies and compounding pharmacies.
79
CA 02725026 2010-12-10
[0252] The kits may include the compounds as described herein packaged in
either a unit
dosage form or in a multi-use form. The kits may also include multiple units
of the unit dose
form. In certain embodiments, provided are the compound described herein in a
unit dose
form. In other embodiments the compositions may be provided in a multi-dose
form (e.g., a
blister pack, etc.).
[0253] The examples below are intended to be purely exemplary of the invention
and
should therefore not be considered to limit the invention in any way. The
following examples
and detailed description are offered by way of illustration and not by way of
limitation.
Additional examples can be found in U.S. Patent Publication No. US
2009/0123419 Al,
which is incorporated herein by reference.
EXAMPLES
Example 1: Phase IB study of 4-iodo-3-nitrobenzamide in combination with
chemotherapy in patients
with advanced solid tumors.
[0254] A Phase IB, open-label, dose escalation study evaluates the safety of 4-
iodo-3-
nitrobenzamide (BA) (2.0, 2.8, 4.0, 5.6, 8.0, and 11.2 mg/kg) in combination
with
chemotherapeutic regimens (topotecan, gemcitabine, temozolomide, and
carboplatin +
paclitaxel) in subjects with advanced solid tumors including ovarian tumors.
The dose-
escalation phase of the study has been completed, and well tolerated
combinations of 4-iodo-
3-nitrobenzamide and cytotoxic chemotherapy have been identified. The protocol
has been
amended to evaluate 4-iodo-3-nitrobenzamide in combination with chemotherapy
in specific
tumor types.
[0255] Rationale. Topotecan targets topoisomerase I, which plays a critical
role in DNA
replication, transcription, and Recombination. Topotecan selectively
stabilizes topoisomerase
I-DNA covalent complexes, inhibiting re-ligation of topoisomerase I-mediated
single-strand
DNA breaks and producing lethal double-strand DNA breaks. Poly(ADP-Ribose)
Polymerase-1 (PARP-1) interacts with topoisomerase I and increases tumor
sensitivity to
topoisomerase 1 inhibitors. Preclinical studies show that the PARP1 inhibitor
4-iodo-3-
nitrobenzamide potentiates the antitumor activity of topotecan. PARP1 is
significantly up-
regulated in human primary ovarian tumors.
[0256] Study Design. 4-iodo-3-nitrobenzamide plus cytotoxic chemotherapy (CTX)
= CTX Dosing:
CA 02725026 2010-12-10
- Topotecan: 1.5 mg/m2 or 1.1 mg/m2 QD for 5 days of 21 day cycle
- Temozolomide: 75 mg/m2 P.O. QD for 21 days of 28 day cycle
- Gemcitabine: 1000 mg/m2 as 30 min. infusion QW; 7 of 8 weeks; initial 28
days for safety
evaluation
- Carboplatin/Paclitaxel: C= AUC of 6; Pxl = 200 mg/m2; both on day 1 of 21
day cycle
= 4-iodo-3-nitrobenzamide Dosing:
- Twice weekly; i.v. infusion
- Standard 3 + 3 design for 4-iodo-3-nitrobenzamide dose escalation
- Dose levels studied: 2.0, 2.8, 4.0, 5.6, 8.0, and up to 11.2 mg/kg
Study Endpoints:
= Safety, tolerability and MTD of each combination
= Clinical response via RECIST every 2 cycles
General Eli ibg ility:
= Subjects >18 years old with a refractory, advanced solid tumor, ECOG PS of
<=
2, and adequate hematological, renal, and hepatic function
= No restriction on number of prior chemotherapeutic regimens
[02571 Efficacy. In terms of efficacy, 53 of 66 subjects demonstrate some
clinical
benefit (Table 1).
Table 1: Clinical Results
'~tukly Arm (N)
l
1 CR - ovarian; 6 PR - 2 breast, 1 uterine, 1 ovarian, 1 renal, 1 sarcoma; 4
SD >= 6 cycles -
1 adenocarcinosarcoma, 1 ACUP, 2 sarcoma; 42 SD >= 2 cycles- multiple tumor
types
[02581 Ovarian cancer patient response. As shown in Figure 1, a patient with
advanced ovarian cancer has a partial response after 4 cycles of 4-iodo-3-
nitrobenzamide in a
81
CA 02725026 2010-12-10
combination with topotecan. Liver lesion (target lesion) shrinks from 4.6 cm
to 1.5 cm. CA
27-29 biomarker also reduces from >300 to <200.
[0259] Preparation of peripheral blood lymphocyte and tumor samples. Whole
blood is collected into EDTA vacutainers and human PBMCs are obtained by BD
VacutainerTM CPTTM Cell Preparation kit according to the manufacturer's
instructions (BD
VacutainerTM, REF 362760). Tumor samples are collected in a sterile container
and placed
immediately on ice. Within 30 minutes, tumor samples are snap-frozen in liquid
nitrogen and
stored at -80 C until homogenized for analysis. The specimen is defrosted on
ice and the wet
weight is documented. The tissue is homogenized using isotonic buffer [7
mmol/L HEPES,
26 mmol/L KCI, 0.1 mmol/L dextran, 0.4 mmol/L EGTA, 0.5 mmol/L MgC12, 45
mmol/L
sucrose (pH 7.8)]. The homogenate is kept on ice throughout the process, and
homogenization is done in 10-second bursts to prevent undue warming of the
sample. Unless
assayed on the day of homogenization, samples are refrozen to -80 C and stored
at this
temperature until analyzed.
[0260] Poly(ADP-ribose) polymerase assay procedure. Cell preparations are
defrosted
rapidly at room temperature and washed twice in ice-cold PBS. The cell pellets
are
resuspended in 0.15 mg/mL digitonin to a density of 1 x 106 to 2 x 106
cells/mL for 5 minutes
to permeabilize the cells (verified by trypan blue staining), following which
9 volumes of ice-
cold isotonic buffer are added and the sample is placed on ice. Maximally
stimulated PARP
activity is measured in replicate samples of 20,000 cells in a reaction
mixture containing 350
mmol/L NAD+ and 10 mg/mL oligonucleotide in a reaction buffer of 100 mmol/L
Tris-HCI,
120 mmol/L MgCl2 (pH 7.8) in a final volume of 100 pL as described in US
Patent
Publication No. US 2009/0123419 Al (which is incorporated herein by reference)
at 26 C in
an oscillating water bath. The reaction is stopped after 6 minutes by the
addition of excess
PARP inhibitor (400 pL of 12.5 pmol/L AG014699) and the cells are blotted onto
a
nitrocellulose membrane (Hybond-N, Amersham) using a 24-well manifold.
Purified PAR
standards are loaded onto each membrane (0-25 pmol monomer equivalent) to
generate a
standard curve and allow quantification. Overnight incubation with the primary
antibody
(1:500 in PBS + 0.05% Tween 20 + 5% milk powder) at 4 C is followed by two
washes in
PBS-T (PBS + 0.05% Tween 20) and then incubation in secondary antibody
(1:1,000 in PBS
+ 0.05% Tween 20 + 5% milk powder) for 1 hour at room temperature. The
incubated
membrane is washed frequently with PBS over the course of 1 hour and then
exposed for 1
minute to enhanced chemiluminescence reaction solution as supplied by the
manufacturer.
Chemiluminesence detected during a 5-minute exposure is measured using a Fuji
LAS3000
82
CA 02725026 2010-12-10
UV Illuminator (Raytek, Sheffield, United Kingdom) and digitized using the
imaging
software (Fuji LAS Image version 1.1, Raytek). The acquired image is analyzed
using Aida
Image Analyzer (version 3.28.001), and results are expressed in LAU/mm2. Three
background areas on the exposed blot are measured and the mean of the
background signal
from the membrane is subtracted from all results. The PAR polymer standard
curve is
analyzed using an unweighted one-site binding nonlinear regression model and
unknowns
read off the standard curve so generated. Results are then expressed relative
to the number of
cells loaded. Triplicate quality control samples of 5,000 L1210 cells are run
with each assay,
all samples from one patient being analyzed on the same blot. Tumor
homogenates are
assayed in a similar manner; however, the homogenization process introduces
sufficient DNA
damage to maximally stimulate PARP activity and oligonucleotide is not
therefore required.
The protein concentration of the homogenate is measured using the BCA protein
assay and
Titertek Multiscan MCC/340 plate reader. Results are expressed in terms of
pmol PAR
formed/mg protein.
[0261] Evaluation of peripheral blood mononuclear cells (PBMCs) from patients
shows
significant and prolonged PARP inhibition after multiple dosing with 4-iodo-3-
nitrobenzamide doses of 2.8 mg/kg or higher (Figure 2).
[0262] Well-tolerated combinations of 4-iodo-3-nitrobenzamide and cytotoxic
chemotherapy are identified. Any toxicities observed are consistent with known
and expected
side effects of each chemotherapeutic regimen. There is no evidence that the
addition of 4-
iodo-3-nitrobenzamide to any tested cytotoxic regimen either potentiates known
toxicities or
increases the frequency of their expected toxicities. A biologically relevant
dose (2.8 mg/kg)
that elicits significant and sustained PARP inhibition at effective
preclinical blood
concentrations is identified. Approximately 80% of subjects demonstrate
evidence of stable
disease for 2 cycles of treatment or more, indicating potential clinical
benefit. The observed
pattern of tumor response is consistent with PARP expression and/or synergy
with
chemotherapeutic agents.
Example 2: Phase II study of 4-iodo-3-nitrobenzamide in combination with
gemcitabine
and carboplatin in platinum-sensitive recurrent ovarian cancer.
[0263] A Phase II trial to evaluate the efficacy of 4-iodo-3-nitrobenzamide
(BA) in
combination with gemcitabine and carboplatin in the treatment of platinum-
sensitive
recurrent ovarian cancer is currently being conducted. Platinum-sensitivity is
defined by
83
CA 02725026 2010-12-10
relapse or recurrence of ovarian cancer six months or more after receiving the
last dose of a
platinum-based chemotherapeutic.
[0264] Primary Endpoint: To evaluate the objective response rate (ORR) of
gemcitabine/carboplatin in combination with 4-iodo-3-nitrobenzamide [ Time
Frame: Until
progressive disease or death ].
[0265] Secondary Endpoints: To determine the nature and degree of toxicity of
gemcitabine/carboplatin in combination with 4-iodo-3-nitrobenzamide [ Time
Frame: 30 days
after last BSI-201 exposure ]; and (2) To evaluate progression-free survival
(PFS) of
gemcitabine/carboplatin in combination with 4-iodo-3-nitrobenzamide [ Time
Frame: until
progressive disease or death ].
[0266] Inclusion Criteria: (1) At least 18 years of age; (2) Histological
diagnosis of
epithelial ovarian carcinoma, fallopian tube cancer, or primary peritoneal
carcinoma; (3)
Completion of only one previous course of chemotherapy which contained a
platinum
therapy, with sensitivity to that regimen. "Platinum-sensitivity" is defined
by a relapse greater
than 6 months after termination of platinum-based chemotherapy; (4) Measurable
disease,
defined by at least one lesion that can be accurately measured in at least one
dimension
(longest dimension to be recorded), and is > 20 mm when measured by
conventional
techniques (palpation, plain x-ray, computed tomography [CT], or magnetic
resonance
imaging [MRI]) or > 10 mm when measured by spiral CT; (5) Adequate organ
function
defined as: absolute neutrophil count (ANC) > 1,500/mm3, platelets >
100,000/mm3,
creatinine clearance > 50mL/min, alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) < 2.5 x upper limit of normal (ULN; or < 5 x ULN in
case of liver
metastases); total bilirubin < 1.5 mg/dL; (6) For women of child bearing
potential,
documented negative pregnancy test within two weeks of study entry and
agreement to
acceptable birth control during the duration of the study therapy; (7) Eastern
Cooperative
Oncology Group (ECOG) performance status 0, 1 or 2; and (8) Signed,
institutional review
board (IRB) approved written informed consent.
[0267] Exclusion Criteria: (1) Concurrent invasive malignancy, not including:
(i) Non-
melanomatous skin cancer; (ii) In situ malignancies; (iii) Concurrent
superficial endometrial
carcinoma, if their endometrial carcinoma is superficial or invades less than
50% the
thickness of the myometrium); (iv) Low risk breast cancer (localized, non-
inflammatory)
treated with curative intent; (v) Lesions identifiable only by positron
emission tomography
(PET); (vi) Prior treatment with poly (ADP-ribose) polymerase (PARP)
inhibitors, including
BSI-201; (vii) Major medical conditions that might affect study participation
(i.e.,
84
CA 02725026 2010-12-10
uncontrolled pulmonary, renal, or hepatic dysfunction, uncontrolled
infection); (vlii) Other
significant co-morbid condition which the investigator feels might compromise
effective and
safe participation in the study, including a history of congestive cardiac
failure or an
electrocardiogram (ECG) suggesting significant conduction defect or myocardial
ischemia;
(ix) Enrollment in another investigational device or drug study, or current
treatment with
other investigational agents; (x) Concurrent radiation therapy to treat
primary disease
throughout the course of the study; (xi) Inability to comply with the
requirements of the
study; (xii) Pregnancy or lactation; and (xiii) Leptomenmgeal disease or brain
metastases
requiring steroids or other therapeutic intervention. The above information is
not intended to
contain all considerations relevant to a patient's potential participation in
a clinical trial.
[0268] A maximum of 41 patients with platinum-sensitive recurrent ovarian
cancer are
being treated in this study using a Simon two-stage design. The primary
endpoint is an
improved overall response rate compared to patients receiving treatment with
gemcitabine
and carboplatin alone determined using historical data from a previous trial.
The secondary
endpoints are improved progression-free survival and patient safety. The
exploratory
endpoints are BRCA status and translational medicine.
[0269] During the first stage, study participants (n=17) are receiving 4-iodo-
3-
nitrobenzamide intravenously at a dose of 5.6 mg/kg on days 1, 4, 8, and 11 of
each cycle,
gemcitabine at a dose of 1000 mg/m2 on days 1 and 8 of each cycle, and
carboplatin at AUC
4 (i.e., at 4 mg/ml-minute) on day 1 of each cycle.
[0270] The first stage of this trial has been closed for interim analysis. The
combination
therapy was well-tolerated, with low grade nausea the most common side effect.
After a
minimum of four cycles/twelve weeks, the overall response rate was 41%, with
10 of 17
patients having stable disease and 7 of 17 in either complete or partial
remission. Median
follow-up will be performed at fifteen weeks. A minimum of 8 responses at the
first stage of
the trial are required before proceeding to the second stage (n=24).
[0271] While preferred embodiments of the present invention have been shown
and
described herein, it will be obvious to those skilled in the art that such
embodiments are
provided by way of example only. Numerous variations, changes, and
substitutions will now
occur to those skilled in the art without departing from the invention. It
should be understood
that various alternatives to the embodiments of the invention described herein
may be
employed in practicing the invention. It is intended that the following claims
define the
scope of the invention and that methods and structures within the scope of
these claims and
their equivalents be covered thereby.
