Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DESCRIPTION
COMBINATION OF CHEMOTHERAPEUTIC COMPOUNDS FOR TREATING
CANCER
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates generally to the fields of cancer and
chemopharmaceuticals. More particularly, it concerns the combined use of
substituted
anthracycline compounds and alkylating agents to treat cancer.
B. Description of Related Art
Cancer is a leading cause of death in most countries, and the result of
billions of
dollars in healthcare expense around the world. Through great effort,
significant advances
have been made in treating cancer, primarily due to the development of
radiation and
chemotherapy-based treatments. Unfortunately, a common problem is tumor cell
resistance to
radiation and chemotherapeutic drugs. For example, NSCLC accounts for at least
80% of the
cases of lung cancer, but patients with NSCLC are geiierally unresponsive to
chemotherapy
(Doyle, 1993). This phenomenon, called pleiotropic drug resistance or multi-
drug resistance
(MDR), may account for much of the drug resistance that occurs in previously
treated cancer
patients.
Oue of the traditional ways to attempt to circumvent this problem of drug
resistance
has been combination chemotherapy. Combination chemotherapy uses the differing
mechanisms of action and cytotoxic potentials of multiple drugs. Commonly used
chemotherapeutic agents are classified by their mode of action, origin, or
structure, although
some drugs do not fit clearly into any single group. The categories include
alkylating agents,
anti-metabolites, antibiotics, alkaloids, and miscellaneous agents (including
hormones).
Agents in the different categories have different sites of action. Although
combination
chemotherapy has been useful in some cases, often times cancer cells becomes
resistant to the
combination of chemotherapeutic agents, thereby reducing the beneficial
effects of a
particular combination.
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SUMMARY OF THE INVENTION
The present invention overcomes the deficiencies in the art by providing
colnpositions
and corresponding methods of treating hyperproliferative diseases such as
cancer in a subject
comprising the coinbined use of an alkylating agent and a substituted
antb.racycline
compound. This coinbination provides a synergistic effect leading to the
effective treatinent
of a variety of different cancers.
In one aspect of the present invention there is disclosed a method of treating
cancer in
a subject comprising administering to the subject in need of the treatinent a
therapeutically
effective amount of an alkylating agent and a substituted anthracycline
compound having the
formula:
1 2
R1
O
H
R4 y2 R3 =
lo
1O
R9
R7 R6 R12
wherein Rl is an alkyl chain, a(-COCH2R13) group, or a C(OH)- C.H2R13) group,
wherein R 13
is: a hydrogen (-H) group; a hydroxyl group (-OH); a methoxy group (-OCH3); an
alkoxy
group having 1-20 carbon atoms; an alkyl group having 1-20 carbon atoms; an
aryl group
having 1-20 carbon atoms; a fatty acyl group having the general structure -O-
CO(CH2)kCH3,
wherein k = an integer from 1-20; or a fatty acyl group having the general
structure -0-CO-
(CH2)L(CH=CH),,,(CH2)õCH3, -O-CO-(CH2)õ-CH2NH2, or -OCO-(CHZ)õCOZH, wherein L
is
an integer between 1-3, m is an integer between 1-6, and n is an integer
between 1-9; R2 and
R3 are independently a hydrogen (-H), a hydroxyl group (-OH), or a methoxy
group (-OCH3);
R4 is a hydrogen (-H) group, a methoxy group (-OCH3), a hydroxyl group (-OH),
or a halide;
Yl and Y2 are independently a double bonded oxygen, sulfur, or nitrogen atom;
Z is a
hydrogen (-H) group, a hydroxy (-OH) group, a-CO2H group, or a-CO2R19 group;
R5 and R6
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are independently a hydrogen (-H) group, a hydroxy (-OH) group, a halide, -
ORI9, -SH, -
SR19, -NH2, -NHR'9, -N(R")2, or -CH3; R9 is a hydrogen (-H) group, -CH3, an
allcyl group, an
aryl group, CHaOH, or CH2F; R10, R" and R12 are independently a hydrogen (-H)
group, a
hydroxy (-OH) group, a halide, -OR19; -SH; -S R19; -NH2; -NHR19; -N(R'9)2 or -
CH3; R19 is an
alkyl chain, an alkylating moiety, a cycloalkyl chain, a cyclic ring, a
hydrogen or a cllain(R)
such as -OCO-(CH2)n-CH2NH2 ; or OCO-(CH2)11-CO2H and its salts; one of R7 and
Rs is a -
H; and one of R7 and R8 is a X-alkyl-aromatic-ring (-XAAR) substituent,
wherein A is an
alkyl group and AR is an unsubstituted phenyl ring, a substituted phenyl ring,
a substituted
five-member ring, a heteroaromatic five-member ring, or a six-member ring
having the
structure:
X~
R18 R14
I
R17 R15
R16
wherein, R14-R18 are independently a hydrogen (-H) group, a hydroxyl (-OH)
group, a
methoxy (-OCH3) group, a nitro (-NO2) group, an amine (-NH2) group, a halide,
an alkoxy
group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an
aryl group
having 1-20 carbon atoms, an alkyl-amino group, an alkyl-thio group, a cyano
group (CN,
SCN), an -CO2H group, or an -CO2R19 group; X is a-O, -N or -S, -SO, or -SO2
group; and A
is (CH2)õ where n = 0-10; wherein if R7 is a XAAR substituent, R8 is not, and
if R8 is a XAAR
substituent, W is not.
In certain aspects, the alkylating agent is temozolomide. The substituted
anthracycline
can have the formula:
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OH
OR
C~CH3 0 '
~
CH3
NH'2: ~ ffC[
The method can further include the human subject previously failing one or
more anti-
glioma therapies. The alkylating agent and the substituted anthracycline
compound can be
administered in a treatment regimen comprising at least one cycle of the
alkylating agent and
the substituted anthracycline compound. The treatment regimen can include
administering
the alkylating agent to the subject multiple times within the treatment cycle.
The treatment
regimen can include administering the substituted anthracycline compound to
the subject
multiple times within the treatinent cycle. The treatment regimen can include
administering
the alkylating agent to the subject prior to administering the substituted
anthracylcine
compound within the treatment cycle. The treatment regimen can include
administering the
alkylating agent to the subject after administering the substituted
anthracylcine compound
within the treatment cycle. The treatment regimen can include administering
the alkylatin'g
agent to the subject prior to and after administering the substituted
anthracylcine compound
within the treatment cycle. The treatment regimen can include administering
the substituted
anthracylcine compound to the subject prior to and after administering the
alkylating agent
within the treatment cycle. The treatinent regimen can include administering
the alkylating
agent at about the same time as administering the substituted anthracycline
compound to the
subject. The cycle can be repeated at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30,
35, 40, or more week intervals for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 20,
or more cycles. In other aspects, the treatment regimen includes administering
temozolomide
at a dose from 75 mg/m2/day to 150 mg/m2/day for 5 days (days 1 to 5 of the
cycle),
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combined with WP744 at a dose from 5 mg/m2/day to 15 mg/m2/day for 3 days
(during days
1 to 5 of the cycle), followed by 14 to 23 days of recovery. In certain
embodiments, the cycle
is repeated at 4 to 6 weelc intervals for a total of 6 to 8 cycles. The
alkylating agent and/or the
substituted anthracycline coinpound can be modified after the first cycle. The
subject can be
evaluated for neurotoxicity and/or ototoxicity during or after the first
cycle. The subject can
be evaluated for blood count and platelet count during and/or after each
cycle. Adininistering
can be through dietary administration, oral administration, or via intravenous
injection. The
oral administration can be in the fonn of a pill or a liquid. The intravenous
injection can be in
the forin of a mixture containing an injectable vehicle. The alkylating agent
can be
administered orally and the substituted antliracylcine compound can be
administered
intravenously. The allcylating agent and the substituted anthracycline
compound can be
included in the same composition. The subject can have previously received
radiation
therapy. The subject can have previously received chemotherapy treatment in
addition to the
alkylating agent and the substituted anthracycline coinpound.
In other aspects, there is disclosed a method of treating cancer comprising
administering to a subject with cancer a therapeutically effective amount of
temozolomide
and a substituted anthracycline compound having the formula:
OT-1
~CH'3 0
~
Nff2 T.CCI
Also disclosed is a method of inhibiting the progression of cancer comprising
administering to a subject with cancer a therapeutically effective amount of
an alkylating
ageiit and a substituted anthracycline compound to inhibit the progression of
cancer. The
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metnoa can be iiu-ther defined as inhibiting the vascularization, growtli or
spread of the
cancer.
In another aspect, there is disclosed a method for inla.ibiting cancer
development
coinprising (a) identifying a subject at risk of developing cancer and (b)
administering to said
subject a dose of an alkylating agent and a substituted anthracycline compound
effective to
ii-Aiibit the development of the cancer. The subject can have a familial
history of cancer or has
been exposed to a carcinogenic enviromnent.
In yet another einbodiment, there is disclosed a method of extending the life
of a
subject having cancer coinprising administering to the subject a
therapeutically effective
amount of an alkylating agent and a substituted anthracycline compound.
Also disclosed is a method of enhancing the effects of temozolomide on cancer
comprising administering to a subject having cancer an amount of a substituted
anthracycline
compound sufficient to enhance the effects of temozolomide on the cancer.
There is also disclosed a method of enhancing the effects of the substituted
anthracycline compound of claim 1 on cancer comprising administering to a
subject having
cancer an amount of temozolomide sufficient to enhance the effects of the
substituted
anthracycline compound on the cancer.
Also disclosed is a method for inhibiting cancer recurrence comprising
administering
to a subject previously having cancer a dose of an alkylating agent and a
substituted
anthracycline compound effective to inhibit the development of the cancer. In
other
aspects, there is disclosed a pharmaceutically acceptable , composition
comprising an
alkylating agent and a substituted anthracycline compound. The alkylating
agent can be
temozolomide.
A kit comprising an alkylating agent and a substituted anthracycline compound
is also
contemplated. The alkylating agent is temozolomide.
It is contemplated that any embodiment discussed in this specification can be
implemented with respect to any method or composition of the invention, and
vice versa.
Furthermore, compositions of the invention can be used to achieve methods of
the invention.
The term "about" or "approximately" are defined as being close to as
understood by
one of ordinary skill in the art, and in one non-limiting embodiment the terms
are defined to
be within 10%, preferably within 5%, more preferably within 1%, and most
preferably within
0.5%.
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'1'he terms "inhibiting," "reducing," or "prevention," or any variation of
these terins,
when used in the claims and/or the specification includes any measurable
decrease or
coinplete inliibition to achieve a desired result.
The tenn "effective," as that term is used in the specification and/or claims,
means
adequate to accoinplish a desired, expected, or intended result.
The terms "patient" or "subject" can include an animal. Preferred animals are
mainmals, including but not limited to huinans, pigs, cats, dogs, rodents,
horses, cattle, sheep,
goats and cows. Preferred patients and subjects are humans.
The use of the word "a" or "an" wllen used in conjunction with the term
"comprising"
in the claims and/or the specification may mean "one," but it is also
consistent with the
meaning of "one or more," "at least one," and "one or more than one."
The use of the term "or" in the claims is used to mean "and/or" unless
explicitly
indicated to refer to alternatives only or the alternatives are mutually
exclusive, although the
disclosure supports a definition that refers to only alternatives and
"and/or."
As used in this specification and claim(s), the words "comprising" (and any
form of
comprising, such as "comprise" and "comprises"), "having" (and any form of
having, such as
"have" and "has"), "including" (and any form of including, such as "includes"
and "include")
or "containing" (and any form of containing, such as "contains" and "contain")
are inclusive
or open-ended and do not exclude additional, unrecited elements or method
steps.
Otlier objects, features and advantages of the present invention will become
apparent
from the following detailed description: It should be understood, however,
that the detailed
description and the examples, while indicating specific embodiments of the
invention, are
given by way of illustration only. Additionally, it is contemplated that
changes and
modifications within the spirit and scope of the invention will become
apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein.
FIG. 1 Ex Vivo Clonogenic Assay in AML Patient Samples. The upper panels
represent the number of blast colonies formed in each of three patients. Data
are depicted in
terms of reduction of absolute numbers of AML colonies. The lower graphs
represent the %
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inhibition of blast colony growth. Closed squares and solid rectangles
represent WP744,
while open circles and open rectangles represent doxorubicin. Each data point
represents the
mean value of duplicate and triplicate measurements +l- SD.
FIG. 2 Survival of Animals Treated with WP744 in a Mouse Model of Glioma.
U87GBM cells were implanted intracranially in nude (Nu-Nu) mice and allowed to
grow for
five days. Animals were then treated with vehicle (PBS) or WP744 10 mg/kg
administered
intravenously A second dose of 5 mg/lcg RTA 744 was administered 10 days after
the first
dose.
FIG. 3 Survival of Animals Receiving Temozolomide +/- WP744 in a Mouse
Model of Glioma. Groups of 6 mice each received vehicle control (PBS, QDx5
i.p.),
teinozolomide (7.5 mg/kg QDx5 i.p.), or teinozolomide (7.5mg/kg QDx5 i.p.)
plus RTA 744
(5 mg/kg, QDx5 i.p.). Median survival in this model was 30 days for the
control group,
compared with 40 days for animals receiving temozolomide alone and 48 days for
animals
receiving the combination of temozolomide plus RTA 744.
FIG. 4. Mean Plasma Concentration vs. Time Curve after Intravenous
Administration of WP744 to Mice. WP744 administered as an intravenous bolus
injection
to CD-1 mice, given at a dose of 20 mg/kg. Each open circle represents data
corresponding to
the mean of 5 mice sampled per time point. The solid line represents the
pharmacokinetic
model fit of the data.
FIG. 5. Tissue Distribution of RTA 744 in Plasma and Brain of CD-1 Mice after
Intravenous Administration (20 mg/kg).
FIG. 6. WP744 Distribution in a Murine Glioma Model. Male tumor-bearing
Nu/Nu mice (U87 xenograft) were given WP744 at 20 mg/kg as an i.v. bolus.
Blood, tumor,
and contralateral brain samples were harvested at 0.5, 1, and 2 hrs post dose
and WP744 was
extracted from plasma (using a solid phase extraction method) and from tissues
(using a
liquid: liquid extraction method).
FIG. 7. Protein binding of WP744 in mouse, rat, dog, and human plasma using
ultrafiltration. WP744 was dissolved in animal plasmas (mouse, rat, dog, and
human) to a
final concentration of 1000 ng/mL. Amicon Centrifree columns were loaded with
1 ml of
treated plasma, placed in fixed-angle centrifuge and centrifuged at 2000 x g
for 30 minutes.
Aliquots (125 ml) were removed from protein-free filtrate, protein-rich
plasma, and unfiltered
plasma, then analyzed for WP744 content.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Drug resistance is a major obstacle in the treatinent of hyperproliferative
diseases such
as cancer. Clinical experience shows that some cancers demonstrate selective
sensitivity to
certain drugs but resistance to others. Treatment decisions, however, are
typically made
empirically using a trial-and-error approach. A need exists for new methods
and therapeutic
compositions that can overcome drug resistance and/or enhance the
effectiveness of other
anti-cancer agents.
The present invention overcome the deficiencies in the art by providing
methods and
compositions for treating hyperproliferative diseases in a subject by
administering a
combination of an alkylating agent and a substituted anthracycline compound
having the
formula as described in this specification to a subject in need of such
treatment. This
combination has a synergistic effect which can lead to more efficient and
efficacious cancer
treatment regimens. These and other aspects of the present invention are
described in further
detail in the following sections.
A. Hyperproliferative Diseases
In certain aspects, the present invention can be used to treatment of
llyperproliferative
diseases including, but not limited to, cancer. A hyperproliferative disease
is any disease or
condition which has, as part of its pathology, an abnormal increase in cell
number. Included
in such diseases are benign conditions such as benign prostatic hypertrophy
and ovarian cysts.
Also included are premalignant lesions, such as squamous hyperplasia. At the
other end of
the spectrum of hyperproliferative diseases are cancers. A hyperproliferative
disease can
involve cells of any cell type. The hyperproliferative disease may or may not
be associated
with an increase in size of individual cells compared to normal cells.
Another type of hyperproliferative disease is a hyperproliferative lesion, a
lesion
characterized by an abnonnal increase in the number of cells. This increase in
the number of
cells may or may not be associated with an increase in size of the lesion.
Examples of
hyperproliferative lesions that are contemplated for treatment include benign
tumors and
preinalignant lesions. Examples include, but are not limited to, squamous cell
hyperplastic
lesions, premalignant epithelial lesions, psoriatic lesions, cutaneous warts,
periungual warts,
anogenital warts, epidermdysplasia verruciformis, intraepithelial neoplastic
lesions, focal
epithelial hyperplasia, conjunctival papilloma, conjunctival carcinoma, or
squamous
carcinoma lesion. The lesion can involve cells of any cell type. Examples
include
keratinocytes, epithelial cells, skin cells, and mucosal cells.
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1. Cancer
"Cancer" includes a tissue of uncontrolled growth or proliferation of cells,
such as a
tuinor. Cancer develops through 'the accuinulation of genetic alterations
(Fearon and
Vogelstein, 1990) and gains a growth advantage over norinal surrounding cells.
The genetic
transformation of normal cells to neoplastic cells occurs througli a series of
progressive steps.
Genetic progression models have been studied in some cancers, such as head and
neck cancer
(Califano et al., 1996). Examples of cancers that can be treated with the
present invention
include, but are not limited to, breast caiicer, lung cancer, prostate cancer,
ovarian cancer,
liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head &
neclc cancer,
bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic
cancer, stomach
cancer, pancreatic cancer, testicular cancer, leukemia, and brain cancer.
2. Brain Cancer
Brain cancer is of particular interest in the context of the present
invention. Brain
cancers include brain tumors (gliomas) which are typically any intracranial
tumor created by
abnonnal or uncontrolled cell division, normally either found in the brain
itself (e.g., neurons,
glia cells (astrocytes, oligodendrocytes, ependymal cells), lymphatic tissue,
blood vessels), in
the cranial neives (myelin-producing Schwann cells), in the brain envelopes
(meninges),
skull, pituitaiy and pineal glands, or spread from cancers primarily located
in other organs
(e.g., metastatic tumors).
Gliomas are a diverse group of brain tumors that arise from the normal "glial"
cells of
the brain. Gliomas have specific signs and symptoms that are primarily related
to the location
of the glioma. The temporal lobe gliomas, for example, may cause epilepsy,
difficulty with
speech or loss of memory. The frontal lobe gliomas may cause behavioral
changes, weakness
of the arms or legs or difficulty with speech. The occipital gliomas may cause
loss of vision.
The parietal gliomas may cause loss of spatial orientation, diminished
sensation on the
opposite side of the body, or inability to recognize once familiar objects or
persons.
The most important determinant of survival for gliomas is the "grade" of the
glioma.
The low-grade gliomas have a protracted natural history, while the high grade
gliomas
(anaplastic astrocytoma and glioblastoina multiforme) are niuch more difficult
to successfully
treat. In this classification, astrocytomas and glioblastomas represent
different grades of
malignancy of the same tumor. Grade I tumors, typically slow growing, are
characterized by
most cells having normal characteristics, and few mitotic features.
Endothelial proliferation is
absent. Grade II tumors, previously designated "astroblastomas," are
characterized by an
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increased number of cells with polyinorphic nuclei in initoses. There is no
clear line of
demarcation from norinal tissue. Grade III tumors represent anaplastic
astrocytomas and
Grade IV tumors represent the typical glioblastoma multiforme, characterized
by cellular
pleomorphisin, vascular proliferation, mitoses, and multinucleated giant
cells.
The current morphologically-based tumor classifications often mix cell lineage
features with tumor growth characteristics. However, there are two general
classifications -
the anaplastic glioma strata and glioblastomas. The former is comprised of
various gliomas
including anaplastic astrocytomas, anaplastic oligoastrocytomas, anaplastic
oligodendroglioinas, malignant glioma, anaplastic gliomas non-specified, and
anaplastic
ependyinoma.
The anaplastic gliomas are intermediate grade infiltrative gliomas -
classified between
low (localized, slow growing) and glioblastoma multiforme (rapidly growing and
highly
invasive). Anaplastic astrocytomas (AA) are tumors that arise from brain cells
called
astrocytes and/or their precursors. Astrocytes are support cells of the
central nervous systein.
The majority of astrocytic tumors in children are low-grade, whereas the
majority in adults are
high-grade. These tumors can occur anywhere in the brain and spinal cord.
Oligodendrogliomas are gliomas derived from oligodendrocytes and/or their
precursors. Oligodendrocytes that have a role in the structure and function of
myelinated
neurons in the brain. Anaplastic oligodendroglioma (AO) are more aggressive
than
oligodendrogliomas, but are also more sensitive to cheinotherapy than are
anaplastic
astrocytomas. A high rate of response to the use of PCV (procarbazine, CCNU,
vincristine)
chemotherapy has led to the common use of PCV cheinotherapy prior to radiation
therapy,
following iiTadiation, and/or at tumor recurrence and progression. Another
glioma appears as
histologic mixture of both oligodendroglioma and astrocytoma tumor forms and
is called
oligoastrocytoma. While oligoastrocytoma can be low-grade, the majority of the
mixed
oligoastrocytomas are anaplastic oligoastrocytomas (AOA).
The last glioma subgroup are ependymomas. One subtype of malignant ependymomas
is the anaplastic ependymoma (AE); these tumors arise from ependymal cells
and/or their
precursors that line the cerebrospinal fluid passageways, called ventricles.
These tumors are
classified as either supratentorial (in the top part of the head) or
infratentorial (in the back of
the head).
Metastatic brain cancer also presents a serious medical challenge. Certain
primary
cancers arising outside the CNS, such as breast and lung cancer, tend to
metastasize to the
brain. Brain metastasis is a frequent cause of mortality in these patients.
Unfortunately, very
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few agents have proven effective in treating CNS metastatic disease. The
anthracyclines of
the current invention, because they have shown activity in preclinical models
of breast cancer
and other non-CNS cancers, and against drug-resistant cancer cell lines, are
useful in treating
metastatic CNS cancer, particularly in coinbination with temozolomide.
The present invention overcomes previous deficiencies in treating
hyperproliferative
diseases such as cancer. The inventor has discovered that the combination of
an alkylating
agent with a substituted anthracycline coinpound has a synergistic treatment
effect. Non-
limiting examples of the alkylating agents and substituted anthracycline
compounds that can
be used in the context of the present invention are described in the following
sections.
B. Alkylating Agents
Alkylating agents include compounds that directly interact with genomic DNA to
prevent the cancer cell from proliferating. These compounds typically have the
ability to 'add
an alkyl group(s) to electronegative groups. They can stop tumor growth by
cross-linking
guanine nucelobases in DNA double-helix strands. This prevents DNA strands
from
uncoiling and separating, thereby preventing DNA replication and cell
division. Non-limiting
examples of aklylating agents that can be used in the context of the present
invention include
temozolomide, busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),
dacarbazine,
ifosfamide, mechlorethamine (mustargen), and melphalan.
In certain aspects, the preferred alkylating agent is temozolomide (3,4-
dihydro-3-
methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide). Temozolomide has been
shown to
treat brain tumors. It is derived from dacarbazine and was first synthesised
in 1984. The
chemical formula for temozolomide is C6H6N602 (CAS # 85622-93-1) and its
corresponding
structure is:
~ ~
~ ~
~
N ~
~' o
\
N N
H2N
Ternozololiiide
Temozolomide is typically administered orally once a day for 5 days in a 28-
day cycle
and is used to treat patients with malignant glioma. It has been shown to have
high
bioavailability and can cross the blood-brain barrier where it is
spontaneously hydrolysed to
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its active form (3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC)).
Teinozolomide is
sold by Schering-Plougli under the brand naine TEMODAR in 5mg, 20mg, 100mg,
and
250ing capsules.
C. Substituted Anthracycline Compounds
Another aspect of the present invention includes using substituted
anthracycline
compounds in coinbination with alkylating agents to treat a variety of
different cancers. Non-
limiting exainples of substituted anthracycline compounds, and methods of
using and making
the same, are described in U.S. Patent 6,673,907 to Priebe et al. ('907
Patent), the disclosure
of which is incorporated by reference in its entirety. In certain aspects, the
`907 Patent
describes two main classes of substituted anthracycline compounds; one bearing
modified
substituents at the C-3' sugar moiety and the otlier bearing modifications at
the C-4' sugar
moiety.
For instance, the substituted anthracycline compound can have the following
generic
formula:
1 2
R1
I I OH
R4 y2 R3 =
O
Rg
5
R7
R6 R12
wherein R' can include any suitable group or combination of groups that form
but are not
limited to a nucleic acid intercalator or binding compound and a topoisomerase
inhibitor,
including but not limited to, an alkyl chain, a(-COCH2R13) group, or a C(OH)-
CH2R13)
group. R13 can be a hydrogen (-H) group, a hydroxyl group (-OH), a methoxy
group
(-OCH3), an alkoxy group having 1-20 carbon atoms, an alkyl group having 1-20
carbon
atoms, an aryl group having 1-20 carbon atoms, a fatty acyl group having the
general structure
-O-CO(CH2)kCH3, wherein k = an integer from 1-20, or a fatty acyl group having
the general
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structure -O-CO-(CH2)L(CH=CH),,,(CH2)õCH3a -O-CO-(CH2)õCH2NH2, or -OCO-(CH2)õ
CO2H, wherein L is an integer between 1-3, m is an integer between 1-6, and n
is an integer
between 1-9. R2 and R3 cati independently be ahydrogen (-H), a hydroxyl (-OH)
group, or a
methoxy (-OCH3) group. R4 can be a hydrogen (-H) group, a methoxy group (-OCHA
a
hydroxyl group (-OH), or a halide. Y' and Ya can independently be a double
bonded oxygen,
sulfur, or nitrogen atom. Z can be a hydrogen (-H) group, a hydroxy (-OH)
group, a-CO2H
group, or a-CO2R19 group. R9 can be a hydrogen (-H) group, -CH3, an alkyl
group, an aryl
group, CH2OH, or CH2F. R10, R" and Rla can independently be a hydrogen (-H)
group, a
hydroxy (-OH) group, a halide, -OR19; -SH; -S Rl9; -NH2; -NHR19; -N(R19)2 or -
CH3. R19 can
be an alkyl chain, an alkylating moiety, a cycloalkyl chain, a cyclic ring, or
a hydrogen.
1. Modified substituents at the C-3' sugar moiety
As noted above, in certain aspects the substituted anthracycline compounds
include
modified substituents at the C-3' sugar moiety. This is exemplified in the
above generic
structure when R6 and R7 can independently be a hydrogen (-H) group, a hydroxy
(-OH)
group, a halide, -OR'9, -SH, -SRig, -NH2, -NHR19, -N(R19)2, or -CH3, and R7
can additionally
be a saccharide. One of R5 and R6 can be a -H and one of R5 and R6 can be an X-
alkyl-
aromatic-ring (-XAAR) substituent, wherein A can be an alkyl group and AR can
be an
unsubstituted phenyl ring, a substituted phenyl ring, a substituted five-
member ring, a
heteroaromatic five-member ring, or a six-member ring having the structure:
X\
A
R18 /I R14
~
R17 R15
R16
wherein Rl4 -Rjg can independently a hydrogen (-H) group, a hydroxyl (-OH)
group, a
methoxy (-OCH3) gr`oup, a nitro (-NOa) group, an amine (-NH2) group, a halide,
an alkoxy
group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an
aryl group
having 1-20 carbon atoms, an alkyl-amino group, an alkyl-thio group, a cyano
group (CN,
SCN), an -CO2H group, or an -CO2R19 group. X can be an -0, -N or -S, -SO, or -
SOZ group.
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A can be (CH2)õ where n = 0-10. In certain aspects, if R5 is a.XAAR
substituent, R6 is not,
and if R6 is a XAAR substituent, R5 is not. Non-limiting examples of
substituted
anthracycline coinpounds that are modified at the C-3' sugar moiety are
described in the `907
Patent at Figures 2-16 as WP831, WP791, WP790, WP787, WP786, WP785, WP784,
WP780, WP778, WP775, WP774, WP758, WP757, WP756, WP755.
2. Modified substituents at the C-4' sugar moiety
In other aspects of the present invention, the substituted anthracycline
compounds
include modified substituents at the C-4' sugar moiety. This is exeinplified
in the above
generic structure when R5 and R6 can independently be a hydrogen (H) group, a
hydroxy (-
OH) group, a halide, -OR19, -SH, -SR19, -NH2, -NHR19, -N(R19)2, or -CH3, and
R5 can
additionally be a saccharide. One of R6 and R7 can be a -H and one of R6 and
R7 can be an X-
alkyl-aromatic-ring (-XAAR) substituent, wherein A can be an alkyl group and
AR can be
an unsubstituted phenyl ring, a substituted phenyl ring, a substituted five-
member ring, a
heteroaromatic five-member ring, or a six-member ring having the structure:
X\
A
Rl8 R14
R17 R15
R16
wherein R14-R18 can independently a hydrogen (-H) group, a hydroxyl (-OH)
group, a
methoxy (-OCH3) group, a nitro (-NO2) group, an amine (-NH2) group, a halide,
an alkoxy
group having 1-20 carbon atoms, an alkyl group having 1-20 carbon atoms, an
aryl group
having 1-20 carbon atoms, an alkyl-amino group, an alkyl-thio group, a cyano
group (CN,
!0 SCN), an -CO2H group, or an -CO2R19 group. X can be an -0, -N or -S, -SO,
or -SO2 group.
A can be (CH2)11 where n= 0-10. In certain aspects, if R7 is a XAAR
substituent, Rg is not,
and if R8 is a XAAR substituent, R7 is not. Non-limiting examples of
substituted
anthracycline compounds that are modified at the C-4' sugar moiety are
described in the `907
Patent at Figares 17-25 as WP799, WP797, WP794, WP783, WP750, WP744, WP727 and
5 WP571.
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In certain einbodiments of the present invention, the preferred substituted
antluacycline coinpound that can be used with the alkylating agents is WP744
which has the
following generic structure:
OR
~~~~ 0 01-T
0
OH~
0
TICI
The chemical name for the above molecule is 4'-O-Benzyl doxorubicin
hydrochloride or 7-0-
(3-amino-4-O-benzyl-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl) adriamycinone
hydrochloride.
Its molecular formula is C34H36C1N011, and molecular weight is 670.1 g/mol.
The
mechanism for action of this compound involves intercalation, disruption of
topoisomerase II
activity, and free radical formation (Denesi et al., 2002; Faderl et al.,
2001).
WP744 can cross the blood brain barrier in amounts sufficient to have a
therapeutic
effect. This characteristic makes WP744 especially useful for treating brain
cancers and
preventing metastases. Non-limiting data shows that this compound has potent
cytotoxic
activity witli some selectivity for cancer cells vs. normal fibroblasts. It is
more potent than
doxorubicin, with a similar spectrum of activity. WP744 demonstrates excellent
in vivo
activity in brain tumors, extending survival in a rigorous U87 orthotopic
mouse model of
glioma. Additionally, activity was demonstrated in the U87 flank tumor model
of glioma, the
mouse L1210 leukemia model and the mouse MDA-MB293 breast cancer xenograft
model.
Further non-limiting data shows that WP744 can surprisingly and unexpectedly
enhance the
effects alkylating agents such as temozolomide.
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D. Treatment Protocol
A tlierapeutic protocol using alkylating agents and substituted anthracycline
compounds in the treatinent of cancer is also conteinplated. For example, the
alkylating
agents can be administered prior to, at the saine time, or after the
substituted anthracycline
coinpounds are administered to the subject within a single cycle. However, it
is contemplated
that otller orders will provide similar results. For example, alkylating agent
is "A" and
substituted anthracycline compound is "B", the following orders can be used:
A/B/A B/A/B A/A/B B/A/A A/B/A/B
B/A/B/A A/A/AB B/A/A/A A/B/A/A A/A/B/A
In addition, it is contemplated that each alkylating agent/substituted
antllracycline
compound can be repeated one, two, three, four, five six, seven, eight, nine,
ten, eleven,
twelve, thirteen, fourteen, or more times. In certain cases, the dosages of
the alkylating agent
and/or substituted anthracycline compound can be adjusted within each cycle or
for each new
cycle.
The following is one example of a particular treatment protocol. A cycle
constitutes 3
to 4 weeks. Temozolomide is administered orally on days 1 througli 5 (150
mg/m2 per day).
WP 744 is administered i.v. (10 ing/m2 per day) on days 1 through 3. The cycle
ends on day
21 to 28, and is then repeated for a total of 6 to 8 cycles, with breaks for
late nadir times and
failure of blood counts to return to an acceptable level within the allotted
14 day period after
coinpletion of a treatment cycle.
In certain non-limiting aspects, a subject can be evaluated by neurological
examination during the study for neurological changes considered to be
independent of tumor
and graded using NCI Common Toxicity Criteria (neurotoxicity). Aside from
baseline
audiometric testing, repeat audiometric testing for ototoxicity can be
performed at the
physician's discretion for patients who had evidence of hearing loss or
progression of hearing
loss by neurological examination. In addition, blood counts can be performed
biweekly, and
serum creatinine, alkaline phosphatase, bilirubin and alanine amino-
transferase tests can be
performed before each cycle.
E. Combination Therapies
In order to increase the effectiveness of a treatment with the compounds of
the present
invention, it may be desirable to combine these compounds other cancer
treatments. Non-
limiting examples of additional treatments that can be used in the context of
the present
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invention include the adininistration of other chemotherapeutic compounds,
radiotlierapy,
iminunotherapy, and surgery.
Non-limiting exainples of coinbination chemotherapies include cisplatin
(CDDP),
carboplatin, procarbazine, mechlorethainine, cyclophosphainide, cainptothecin,
ifosfamide,
meiphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,
doxorubicin,
bleomycin, plicomycin, initomycin, etoposide (VP16), tamoxifen, raloxifene,
estrogen
receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein
transferase inhibitors,
transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate,
Temazolomide (an
aqueous fonn of DTIC), or any analog or derivative variant of the foregoing.
As for radiotherapy, non-limiting examples include the use of y-rays, X-rays,
and/or
the directed delivery of radioisotopes to tumor cells. Other forms of DNA
damaging factors
are also contemplated such as microwaves and UV-irradiation. It is most likely
that all of
these factors effect a broad range of damage on DNA, on the precursors of DNA,
on the
replication and repair of DNA, and on the assembly and maintenance of
chromosonies.
Dosage ranges for X-rays can range from daily doses of 50 to 200 roentgens for
prolonged
periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage
ranges for
radioisotopes can also vary, and often times depend on the half-life of the
isotope, the strength
and type of radiation emitted, and the uptake by the neoplastic cells.
Iminunotherapeutics, generally, rely on the use of iminune effector cells and
molecules to target and destroy cancer cells. The immune effector may be, for
example, an
antibody specific for some marker on the surface of a tumor cell. The antibody
alone may
serve as an effector of therapy or it may recruit other cells to actually
effect cell killing. The
antibody also may be conjugated to a drug or toxin (chemotherapeutic,
radionucleotide, ricin
A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting
agent.
Alternatively, the effector may be a lymphocyte carrying a surface molecule
that interacts,
either directly or indirectly, with a tumor cell target. Various effector
cells include cytotoxic
T cells and NK cells.
Surgery can include resection in which all or part of cancerous or other
relevant tissue
is physically removed, excised, and/or destroyed. Tumor resection includes
physical removal
of at least part of a tumor. In addition to tumor resection, treatment by
surgery includes laser
surgery, cryosurgery, electrosurgery, and microscopically controlled surgery
(Mohs' surgery),
laparascopic surgery and harmonic scalpel surgery.
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F. Modifications and Equivalents
It is also contemplated that modifications can be made to the alkylating
agents and
substituted anthracycline coinpounds of the present invention. Non-limiting
examples of such
modifications include the addition or removal of lower alkanes such as
inetliyl, etliyl, propyl,
or substituted lower alkanes such as hydroxyinethyl or aininoinethyl groups;
carboxyl groups
and carbonyl groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate,
sulfonate,
sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate, phosphono, phosphoryl
groups, and halide
substituents. Additional modifications can include an addition or a deletion
of one or more
atoms of the atomic frameworlc, for example, substitution of an ethyl by a
propyl; substitution
of a phenyl by a larger or smaller aromatic group. Alternatively, in a cyclic
or bicyclic
structure, hetero atoms such as N, S, or 0 can be substituted into the
structure instead of a
carbon atom.
Known and unknown equivalents to the compounds discussed throughout this
specification can be used with the compositions and methods of the present
invention. The
equivalents can be used as substitutes for the specific compounds, agents, and
active
components. The equivalents can also be used to add to the methods and
compositions of the
present invention. A person of ordinary skill in the art would be able to
recognize and
identify acceptable known and unknown equivalents to the specific compounds,
agents, and
active ingredients without undue experimentation.
G. Compositions and Routes of Administration
One embodiment of this invention includes methods of treating or preventing
cancer
by administering to a subject a coinbination of an alkylating agent and a
substituted
anthracycline compound as described throughout this specification. The
following
subsections provide non-limiting examples of pharmaceutical coinpositions and
routes of
administration.
1. Compositions
Compositions of the present invention can include an alkylating agent or a
substituted
anthracycline compound, or both. The phrases "pharmaceutical or
pharmacologically
acceptable" refers to molecular entities and compositions that do not produce
an adverse,
allergic or other untoward reaction when administered to an animal, such as,
for example, a
human. The preparation of a pharmaceutical composition(s) will be known to
those of skill in
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the art in light of the present disclosure, as exemplified by Remington's
Pharmaceutical
Sciences, 18th Ed. Mack Priiiting Company, 1990.
"Therapeutically effective amounts" include amounts effective to produce a
beneficial
result in the recipient subject. For instance, an effective amount can include
an amount
sufficient to detectably and/or repeatedly ameliorate, reduce, minimize or
limit the extent of
the disease or its symptoms. More rigorous definitions may apply, including
elimination,
eradication or cure of disease. Such amounts inay be initially determined by
reviewing the
published literature, by conducting ifz vitro tests, or by conducting
metabolic studies in
healthy experimental animals. Before use in a clinical setting, it may be
beneficial to conduct
confirmatory studies in an animal model, preferably a widely accepted animal
model of the
particular disease to be treated. Preferred animal models for use in certain
embodiments are
rodent models, which are preferred because they are economical to use and,
particularly,
because the results gained are widely accepted as predictive of clinical
value.
Non limiting examples of "pharmaceutically acceptable carrier" include
solvents,
dispersion media, coatings, surfactants, antioxidants, preservatives (e.g.,
antibacterial agents,
antifungal ageilts), isotonic agents, absorption delaying agents, salts,
preservatives, drugs,
drug stabilizers, gels, binders, excipients, disintegration agents,
lubricants, sweetening agents,
flavoring agents, dyes, such like materials and combination s thereof, as
would be known to
one of ordinary skill in the art (Remington's, 1990). Except insofar as any
conventional
carrier is incompatible with the active ingredient, its use in the therapeutic
or pharmaceutical
compositions is contemplated.
The actual dosage amount of active ingredients of the present invention (e.g.,
alkylating agents and substituted anthracycline coinpounds) administered to a
subject can be
determined by physical and physiological factors such as body weight, severity
of condition,
the type of disease being treated, previous or concurrent therapeutic
interventions, idiopathy
of the patient and on the route of administration. The practitioner
responsible for
administration can determine the concentration of ingredient in a composition
and appropriate
dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example,
at
least about 0.1% of an active coinpound. In other embodiments, the an active
compound may
comprise between about 2% to about 75% of the weight of the unit, or between
about 25% to
about 60%, for example, and any range derivable therein. In other non-limiting
examples, a
dose may also comprise from about 1 microgram/kg/body weight, about 5
microgram/kg/body weight, about 10 microgram/kg/body weight, about 50
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microgram/kg/body weight, about 100 micrograin/lcg/body weight, about 200
inicrograin/lcg/body weight, about 350 microgram/lcg/body weigllt, about 500
microgram/kg/body weight, about 1 milligrain/lcg/body weight, about 5
milligrain/lcg/body
weight, about 10 inilligram/lcg/body weight, about 50 inilligrain/lcg/body
weight, about 100
milligram/kg/body weight, about 200 milligram/lcg/body weiglit, about 350
inilligram/lcg/body weight, about 500 milligram/kg/body weight, to about 1000
mg/kg/body
weight or more per administration, and any range derivable therein. In non-
limiting exainples
of a derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to
about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500
milligram/kg/body weight, etc., can be administered, based on the nuinbers
described above.
Alternatively, a subject may be given 1 x 10'5, 10-6, 10-6, 10'7 , 10-8, 10'9
, 10-10,
10-11, 10-12 M of a substance (or any range derivable therein), of an active
ingredient, in a
volume of 0.1 1, 1.0 1, 10 1, 100 1, 1 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50 ml,
100 ml, 200 ml, 300
ml, 400 ml, 500 ml, or more (or any range derivable therein). Active
ingredients may be
administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times over a course of 1,
2, 3, 4, 5, 6, 7, 8, 9
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4,
5, 6, 7 days, 1, 2, 3,
4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more
years on a regular or as needed basis.
The composition can also include various antioxidants to retard oxidation of
one or
more active ingredients. Additionally, the prevention of the action of
microorganisms can be
brought about by preservatives such as various antibacterial and antifungal
agents, including
but not limited to parabens (e.g., methylparabens, propylparabens),
chlorobutanol, phenol,
sorbic acid, thimerosal or combinations thereof.
The compositions of the present invention can include different types of
carriers
depending on whetller it is to be administered in solid, liquid or aerosol
form, and whether it
need to be sterile for such routes of administration as injection.
The compositions may be formulated into a composition in a free base, neutral
or salt
fonn. Pharmaceutically acceptable salts, include the acid addition salts,
e.g., those formed
with the free amino groups of a proteinaceous composition, or which are formed
with
inorganic acids such as for example, hydrochloric or phosphoric acids, or such
organic acids
as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free
carboxyl groups can
also be derived from inorganic bases such as for example, sodium, potassium,
ammonium,
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calcium or ferric hydroxides; or sucll organic bases as isopropylamine,
triinethylamine,
histidine or procaine.
In einbodiinents where the composition is in a liquid form, a carrier can be a
solvent or
dispersion medium comprising but not limited to, water, ethanol, polyol (e.g.,
glycerol,
propylene glycol, liquid polyetliylene glycol, etc), lipids (e.g.,
triglycerides, vegetable oils,
liposoines) and combinations thereof. The proper fluidity can be maintained,
for exainple, by
the use of a coating, such as lecithin; by the maintenance of the required
particle size by
dispersion in carriers such as, for example liquid polyol or lipids; by the
use of surfactants
sucli as, for example hydroxypropylcellulose; or combinations thereof such
methods. In
many cases, it will be preferable to include isotonic agents, such as, for
example, sugars,
sodium chloride or combinations thereof.
In certain embodiments, the compositions are prepared for administration by
such
routes as oral ingestion. In these einbodiments, the solid composition may
comprise, for
example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g.,
hard or soft shelled
gelatin capsules), sustained release formulations, buccal compositions,
troches, elixirs,
suspensions, syrups, wafers, or combinations thereof. Oral compositions may be
incorporated
directly with the food of the diet. Examples of carriers for oral
administration comprise inert
diluents, assimilable edible carriers or combinations thereof. In other
aspects of the invention,
the oral composition may be prepared as a syrup or elixir. A syrup or elixir,
and may
coinprise, for example, at least one active agent, a sweetening agent, a
preservative, a
flavoring agent, a dye, a preservative, or combinations thereof.
In certain aspects, an oral composition may comprise one or more binders,
excipients,
disintegration agents, lubricants, flavoring agents, and combinations thereof.
Binders can
include, for example, gum tragacanth, acacia, cornstarch, gelatin or
combinations thereof.
Excipients can include, for example, dicalcium phosphate, mannitol, lactose,
starch,
magnesiuin stearate, sodium saccharine, cellulose, magnesium carbonate or com
binations
thereof. Disintegrating agenta can include, for example, corn starch, potato
starch, alginic
acid or combinations thereof. Lubricants can include, for example, magnesium
stearate.
Sweetening agents can include, for example, sucrose, lactose, saccharin or
combinations
thereof. Flavoring agents can include, for example, peppermint, oil of
wintergreen, cherry
flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it
may contain, in
addition -to materials of the above type, carriers such as a liquid carrier.
Various other
materials may be present as coatings or to otherwise modify the physical form
of the dosage
unit. For instance, tablets, pills, or capsules may be coated with shellac,
sugar or both.
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Additional formulations which are suitable for other modes of administration
include
suppositories. Suppositories include solid dosage forins of various weiglits
and shapes,
usually medicated, for insertion into the rectuin, vagina or urethra. After
insertion,
suppositories soften, melt or dissolve in the cavity fluids. In general, for
suppositories,
traditional carriers may include, for example, polyalkylene glycols,
triglycerides or
coinbinations thereof.
Sterile injectable solutions can be prepared by incorporating the active
coinpounds in
the required amount in the appropriate solvent with various of the other
ingredients
enumerated above followed by filtered sterilization. Generally, dispersions
can be prepared
by incorporating the various sterilized active ingredients into a sterile
vehicle which contains
the basic dispersion medium and/or the other ingredients. In the case of
sterile powders for
the preparation of sterile injectable solutions, suspensions or emulsion, the
preferred methods
of preparation are vacuum-drying or freeze-drying techniques which yield a
powder of the
active ingredient plus any additional desired ingredient from a previously
sterile-filtered
liquid medium thereof. The liquid medium should be suitably buffered if
necessary and the
liquid diluent first rendered isotonic prior to injection with sufficient
saline or glucose. The
preparation of highly concentrated compositions for direct injection is also
contemplated,
where the use of DMSO as solvent is envisioned to result in extremely rapid
penetration,
delivering high concentrations of the active agents to a small area.
2. Routes of Administration
The present invention can be administered intravenously, intradermally,
intraarterially,
intraperitoneally, intralesionally, intracranially, intraarticularly,
intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally, intravaginally,
intrauterinely,
intrarectally, topically, intratumorally, intramuscularly, intraperitoneally,
subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically,
intraocularally, orally, topically, locally, inhalation (e.g.. aerosol
inhalation), injection,
inf-usion, continuous infusion, localized perfusion bathing target cells
directly, via a catheter,
via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other
method or any
combination of the forgoing as would be known to one of ordinary skill in the
art
(Remington's, 1990).
H. Kits
The inventor also contemplates the use of a kits in certain aspects of the
present
invention. For example, any of the compositions, compounds, agents, or
ingredients
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described in this specification may be included in a kit. In a non-limiting
exainple, a kit can
include an alkylating agent or a substituted antliracycline coinpound, or
both. The alkylating
agent and/or the substituted anthracycline coinpound can be included into
separate
coinpositions or the same composition.
Where there is more than one coinponent in the kit (they may be packaged
together),
the kit also will generally contain a second, third or other additional
containers into which the
additional components may be separately placed. The kits of the present
invention also can
include a container housing the components in close confinement for commercial
sale. Such
containers may include injection or blow-molded plastic containers into which
the. desired
bottles, dispensers, or packages are retained.
A kit can also include instructions for employing the kit coinponents as well
the use of
any other compositions, compounds, agents, ingredients, or objects not
included in the kit.
Instructions may include variations that can be implemented. For example, the
instructions
can include an explanation of how to apply, use, and maintain the products or
compositions.
EXAMPLES
The following examples are included to demonstrate certain non-limiting
aspects of
the invention. It should be appreciated by those of skill in the art that the
techniques disclosed
in the examples which follow represent techniques discovered by the inventor
to function well
in the practice of the invention. However, those of skill in the art should,
in light of the
present disclosure, appreciate that many changes can be made in the specific
embodiments
which are disclosed and still obtain a like or similar result without
departing from the spirit
and scope of the invention.
EXAMPLE 1
In Vitro Pharmacology Studies
In Vitro Cytotoxicity: WP744 is more potent than doxorubicin in a panel of
human tumor cell lines (Table 1). Human tumor cells were plated on Day 0 in 96-
well plates
with each condition replicated six times. Cells were treated with 10-fold
serial dilutions (1
M to 0.1 nM) of WP744 or doxorubicin on Days I and 3. Control cells were
treated with
vehicle (DMSO) alone. The cells were fixed on Day 5 and stained with
sulforhodamine B.
Fractional survival was detennined by dividing the average absorbance (A492)
value of test
wells by control wells. The fractional survival values were plotted against
the log [drug
concentration] to determine the IC50 value for each drug and cell line (Table
1). In the broad
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panel of solid tumor cell lines tested, WP744 was more potent than doxorubicin
at inhibiting
cell growth.
Table 1. IC50 Values for WP744 and Doxorubicin in Various Human Tumor Cell
Lines
Cell Line Tumor Type WP744 IC50 (nM) Doxorubicin IC50 (nM)
MCF-7 Breast 0.72 4.02
NCI-H522 Lung 3.40 8.32
A549 Lung 1.07 4.68
NCI-H23 Lung 1.73 2.61
SW480 Colon 2.99 8.26
HT-29 Colon 3.66 23.40
AsPC-1 Pancreas 5.62 80.50
BxPC-3 Pancreas 4.05 15.70
Capan-1 Pancreas 5.30 30.75
OVCAR-3 Ovarian 5.31 11.53
Ex Vivo Cytotoxicity in AML Bone Mat row Cells: The cytotoxic potential of
WP744
was further examined in bone marrow cells obtained from three patients with
AML. After
fractionation of adherent cells and depletion of T lymphocytes, remaining
cells were cultured
for clonogenic assay with WP744 and doxorubicin at concentrations ranging from
0.05 to 0.5
gg/mL. Both WP744 and doxorubicin consistently inhibited proliferation of AML
blast
colony-forming cells in a dose-dependent manner (FIG. 1). Patient samples
(particularly from
patients #2 and #3) were substantially more sensitive to RTA 744 than to
doxorubicin.
In Vitro Selectivity: WP744 caused DNA fragmentation in CEM leukemia cells at
one-tenth the concentration needed by doxorubicin to induce the same degree of
damage. In
contrast, WP744 requires a 10-fold higher concentration to cause similar
damage in normal
fibroblasts (Table 2).
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Table 2. Apoptotic Fragmentation Induced by WP744 in CEM Leukemia Cells
versus Doxorubicin and in Normal W138 Fibroblasts
WP744 Doxorubicin WP744
% Fragmented DNA % Fragmented DNA % Total DNA
Concentration ( M) CEM Leukemia CEM Leukemia W138 Fibroblasts
0 0 0 0
0.05 9.5
0.1 31.0 0.2
0.5 50.6 13.9 6.5
1 28.5
2 42.3 41.1 6.5
42.4 55.5
Note: Values are fragmented DNA expressed as percentage of total DNA, as
determined by
quantitative apoptotic fraginentation assay of CEM leulcemic cells incubated
with drugs for 24 hrs.
Data Source: Faderl S, Estrov Z, Kantarjian HM, et al: WP744, a novel
anthracycline with
enhanced proapoptotic and antileukernic activity. Anticancer Res 21: 3777-84,
2001
EXAMPLE 2
5 In Vivo Pharmacology Studies of WP744 Alone and In Combination With
Temozolomide
Mouse Glioma Orthotopic Model: Two studies of WP744 as a single agent and one
study of WP744 in combination with temozolomide were conducted in an
orthotopic mouse
model of glioblastoma multiforme. In this model, nude mice were seeded with
U87GBM
cells by direct intracerebral injection. This places the tumors in the natural
setting and requires
compounds to cross the blood-brain barrier in order to be effective. Tumors
are allowed to
develop over 5 days prior to treatment.
Treatment with WP744 extended survival by 33% compared to vehicle controls
(FIG.
2). Similar results were seen in a second study testing different dosing
regimens in this saine
model. WP744 was administered using 7 mg/kg 5 days on 4 days off, 17.5 mg/kg
twice
weekly. Compared to controls, survival was extended significantly by both
regimens.
A third study demonstrated that the combination of WP744 with temozolomide
produced better survival than vehicle or temozolomide alone (FIG. 3). Median
survival
increased 60% over controls for the combination group, compared with 33 % for
animals
receiving temozolomide alone. Additionally, one animal in the combination
therapy group
survived to day 78, whereas control and temozolomide-treated animals were all
dead by days
32 and 45, respectively.
Mouse U87 Flank Tumor Model: The in vivo activity of WP744 in brain tumors was
further characterized using a flank tumor model of glioma. Mice (Nu-Nu) were
implanted
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with U87 tumor xenografts by subcutaneous injections into the flank and then
treated with
WP744 or teinozolomide intraperitoneally as indicated in Table 3. WP744
deinonstrated a
consistent, dose-dependent effect, iiihibiting tumor growth by as mucli as
65%.
Table 3. TGI for WP744 or Temozolomide Treatment - U87 Flank Tumor
Total
Group Treatment (Dose) Dosing Days Dose Max Weight Change TGI
(mg/kg)
1 Vehicle Control 1-5 3.9% 0.00%
2 WP744 (7 mg/kg) 1-5 and 10-14 70 -20.8% 65.69%
3 WP744 (15 mg/kg) 1, 5, 9 and 13 60 -14.0% 65.11%
4 WP744 (17.5 mg/kg) 1, 6 and 11 52.5 -15.4% 58.52%
Temozolomide (137 mg/kg) 1-5 685 -15.3% 81.75%
Note: 8 animals were treated per group. Treatment was started when the average
tumor size was - 72 mg.
5
MDA-MB-231 Breast Cancer Model: To further characterize the in vivo activity
of
WP744 and compare it to the standard agent doxorubicin, WP744 was evaluated in
the MDA-
MB-231 human breast cancer xenograft mouse model. WP744 and doxorubicin were
administered i.p. to 7 animals per group on the schedules listed in Table 4.
Treatment was
started when the average tumor size was approximately 40 mg. WP744 exhibited a
dose-
dependent anti-tumor effect in this relatively treatment-resistant model
(Table 4). The
doxorubicin group performed slightly better than the WP 744 15 mg/kg group;
however, this
difference was not statistically significant and there was one animal death in
this group
compared with none in the WP744-treated groups.
Table 4. Tumor Weights after Treatment with WP744 or
Doxorubicin in the MDA-MB-231 Breast Cancer Model
Tumor Weight (mg)
Group Treatment (dose) Dosing Days Initial Final %TGI
1 Vehicle 1-5 38.4 1141 0.00
2 WP744 5 m/k 1-5, 7, 8, 10, 12, 14 and 16-19 38.1 1017 10.2
3 WP744 6 mg/kg) 1-5, 8, 10, 12, 14 and 16-19 38.4 987 13.5
4 WP744 (15 mg/kg) 1, 3, 10 and 14 38.4 866 24.1
5 Doxorubicin 3 mg/kg) 1-5 34.7 714 30.8
~%TGI = 100 * (1-(Tx End / Tx Begin)/(Ctrl End / Ctrl Begin))
EXAMPLE 3
Preelinical Pharmacokinetics
Preclinical pharmacokinetic studies were performed in CD-1 mice. Blood samples
(0.5 ml) were collected in heparinized tubes from groups of five mice at
selected time points
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up to 72 hours following drug adininistration. Based on these results, the
intravenous
pharmacokinetics of WP744 in the mouse are best described using a two
coinpartinent model
(FIG. 4). Suminary data are presented in Table 5.
Table 5. Summary of Intravenous Pharmacokinetics in the Mouse
WP744 Doxorubicin ,
Dose (mg/kg) 20 10
Cmax (ng/mL) 907.4 173.9 950
AUCo_. (ng*hr/mL) 2741 237
Vd (L/kg) 22.0 4.2 15.2 (10)
T1/2a, (hr) 1.08 0.25 0.067 (67)
T112p (hr) 12.6 1.7 7.3 (24.6)
CL (L/hr/kg) 7.30 0.63 4 08 (11 0)
Values are expressed as the mean SD
2 Values are expressed as the mean and coefficient of variation (CV)
3 Data taken from Gustafson et. al. 2002
EXAMPLE 4
Preclinical Distribution and Metabolism
In Vivo Biodistribution: The tissue distribution of WP744 after intravenous
administration was studied in CD-I mice. WP744 crossed the blood-brain barrier
and reached
high concentrations in brain tissue at 1 hour following a single i.v. bolus
injection (FIG. 5).
Mean maximum brain concentration at 1 hour was 244.3 ng/g compared to a plasma
concentration of 374.1 ng/mL. The distribution of WP744 into brain tissue
after intravenous
administration was also studied in a murine glioma model (FIG. 6). WP744
sequestered
preferentially in the tuinor tissue versus brain tissue. At 1 hour following
administration of
WP744, the concentration of WP744 was approximately 10-fold higher in the
tumor tissue
than contralateral brain tissue.
In Vitro Protein Binding: Ultrafiltration methods were employed to assess the
relative
in vitro plasma protein binding affinity of WP744. Protein binding varied
between species
with the relative order of binding: human > rat > dog > mouse (FIG. 7).
Metabolic Pr=ofile of WP744: In vitro drug metabolism studies of WP744 were
conducted in the murine S9 liver fraction model. After the activity of the
test system was
confirmed, WP744 was added to the samples and reactions were stopped with
acetonitrile at
4, 7, and 24 hour time points. Extracted samples were subjected to HPLC
analysis and UV
detection using a photodiode array detector. Spectra extracted from these
analyses
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demonstrated the time dependent formation of a more hydrophilic metabolite,
which elutes
from this system 3 minutes prior to the elution of the precursor parent
compound WP744.
To identify and further characterize the WP744 metabolite, quadrupolar time-of-
flight
mass spectroinetry was einployed. Results from this analysis illustrate a +2
reduction of
WP744 indicating that the principal metabolite, as with doxorubicin, is most
probably a two-
electron reduction of the side cliain carbonyl group to a secondary alcohol,
yielding a 4'-O-
benzyl doxorubicinol.
EXAMPLE 5
Clinical Pharmacolcinetics
The pharmacokinetic profile of WP744 injection in humans is being ascertained
in a
current Phase 1 dose escalation clinical trial. In this trial, WP744 injection
is administered to
patients as a daily 2-hour intravenous infusion for three consecutive days.
Blood samples are
taken at pre-specified time points on days 1 through 5 and analyzed for RTA
744
concentration. Data are available for four patients treated with WP744. Based
on these four
patients, the half-life of WP744 is greater than 24 hours (Table 6).
Table 6. Pharmacokinetics of WP744 Injection in Humans
Parameter Patient 101 Patient 102 Patient 103 Patient 104
Dose Level 1.2 mg/m2/day x 2.4 mg/m2/day x 2.4 mg/m2/day x 2.4 mg/mZ/day x
3 days 3 days 3 days 3 days
Cmax (ng/mi) 1.50, 1.39, 1.70 3.42, 3.60, 4.24 2.66, 3.41, 6.15 5.75, 4.03,
8.69
Clearance 39 106 37.3 27.6
(L/hr/m2)
T1i2p (hrs) 33.4 21.7 46.7 38.2
Vss (L/m2) 1550 2817 2287 1366
EXAMPLE 6
Preclinical Toxicology Studies
WP744 has been studied in a number of preclinical toxicology studies,
including IND-
directed GLP Toxicology studies in rats and dogs. Based on these studies,
target organs of
toxicity appear to be the bone marrow, lymphoid organs, digestive tract, and
heart. Although
WP744 has been shown to cross the blood-brain barrier, no signs of
neurotoxicity have been
observed during preclinical studies in any species, and histopathology
revealed no primary
drug-related effects on the central nervous system. The overall pattern of
toxicity of WP744
appears consistent with that of other anthracyclines, including doxorubicin
and epirubicin.
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Single Dose Toxicity Studies: Acute toxicity studies of a single intravenous
dose of
WP744 were conducted in CD-1 mice and Fisher 344 and Sprague-Dawley rats.
Findings
from these studies are suirnnarized in Table 7.
Table 7
Species/Strain/ Doses Tested
Gender (mg/kg) Results Comments
Mouse/ CD-1/ 0, 15, 20, 25 MTD = 40 mg/kg Morbidity observed 5 days following
Male 30, 40, 50, 60 administration of 50 mg/lcg. Mortality
per group occurred on day 6 at 60 mg/kg.
Mouse/ CD-1/ 0, 40, 50, 60, Upper bracket dose = 70 One death occurred at 40
mg/kg, but due
Female 70, 80, 90, 100 mg/kg to a lack of mortality at 50 mg/kg, the
5 per group Lower bracket dose = 50 latter was selected as the lower bracket
mg/kg dose.
Mouse/ CD-1/ 0, 51, 54, 56, MTD < 51 mg/kg Morbidity and mortality observed in
all
Female 59, 62, 65, 69 treatment groups.
per group
Rat/ Fisher 344/ 5, 10, 20, 30, MTD = 20 mg/kg Mortality and morbidity in all
animals
Female 40,'50 with single intravenous dose>_30 mg/kg
4 per group beginning on day 5.
Rat/ Sprague- 0, 5, 10, 15, 20, Females: MTD = 10 mg/kg Evaluation of overall
dose tolerance,
Dawley/ Male & 30, 40, 50 Males: MTD = 15 mg/kg based on a more global
assessment of
Female 5 per gender toxicity, including weight loss, extent of
per group weight loss, weight recovery, time to
weight recovery, and mortality at doses
up to 20 mg/kg suggests that females
were slightly more tolerant of higher
WP744 doses.
5
Multiple Dose Toxicity Studies: WP744 has been studied in multiple dose
toxicity
studies in CD-1 and nu/nu mice, Sprague-Dawley rats, and Beagle dogs. 28-day
GLP
Toxicology studies of WP744 administered intravenously for three consecutive
days were
completed in Sprague-Dawley rats and beagle dogs. Findings from these studies
are
10 summarized in Table 8.
Table 8
Species/
Strain/ Duration
Gender of Study Dose/Schedule Results Noteworthy Findings
Mouse 3 weeks 0, 8, 12, 16, 20 MTD = 16 mg/kg Mortality and morbidity were
observed in 7 of 20
/CD-1/ mg/kg twice/wk once /wk animals following 3 to 6 doses of 8 mg/kg/dose
twice
Male 16 mg/kg once/ wk MTD <8 mg/kg weekly and in 1 of 20 animals receiving 16
mg/kg once
Administered i.v.. twice/ wk weekly. Mortality and morbidity were observed in
all
15-20 per group animals in the 12, 16 and 20 mg/kg/dose groups after 3 to
4 doses given twice weekly.
Heart, bone marrow and splenic red pulp (hematopoietic
tissue) were target organs of toxicity for WP744. The
bone marrow and splenic red pulp atrophy appear to be
recoverable within 2 weeks. Persistent modest to mild
vacuolation in the heart was observed following both
treatment schedules at 2 and 6 weeks post administration.
Mouse 4 weeks 6, 8, 10, 12 mg/kg > 12 m/k once/ Intravenous doses of 6 to 12
mg/kg WP744 are acutely
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Species/
Strain/ Duration
Gender of Study Dose/Scheclule Results Notewortliy rindin Ts
/CD-1/ once/w(c wk tolerated once weekly (total cuintilative doses of 24-48
Female 0, 6, 8, 10, 12 8 mg/kg twice/ wk mg/kg over 4 weeks). Morbidity anci
mortality are
mg/kg twice/wk observed at doses above 8 mg/kg/dose twice weekly.
Aclministered i.v.
per rou
Mouse/ 26 days 6, 7, 8, 9 mg/kg MTD (qdx5) = 7 qdx5 Schedule: Animals treated
with two cycles at 6 or 7
nu/nu/ daily x 5 mg/kg mg/kg demonstrated moderate but not significant weight
Female 15, 20, 25, 30 loss. However, two cycles of 8ing/lcg WP744 resulted in
mg/kg every 4 days MTD (q4d) < 15 significant weight loss and deaths.
15, 20, 25, 30, 35 mg/kg c~4d Schedule: Animals treated with up to 6 cycles of
mg/kg every 5 days WP744 at doses between 15-30 ing/kg demonstrated
MTD (q5d) = 15 dose-dependent toxicity with the 15 mg/kg dose resulting
Administered i.p. mg/kg in 3/5 deaths before study completion, thereby
exceeding
MTD for this schedule.
5 per group q5d Schedule: No deaths were reported in the 15 mg/kg
group although significant weight loss (28%) was noted
at study end. Mortality and significant weight loss
occurred in groups dosed with 20-35 mg/kg WP744.
Rat/ 28 days Phase 1: NOAEL = 2 mg/kg In the 2 mg/kg group, there were no drug-
related deaths.
Sprague- 0 mg/kg daily x 3 qdx3 High mortality was observed in the 5 mg/kg and
8 mg/kg
Dawley/ 2 mg/kg daily x 3 groups on days 8 to 13. As a result, Part 2 of the
study
5 mg/kg daily x 3 was initiated with additional dose groups. No deaths
8 mg/kg daily x 3 occurred in the.8 mg/kg dose group. 5/20 males and
4/20 females in the 3 mg/kg group were found dead or
Phase 2: euthanized moribund
0 daily x 3 Clinical observations included weight loss, decreased
0.8 daily x 3 activity, inucoid stools, rough fur coat, dehydration,
3 daily x 3 facial edema, and dyspnea.
Myelosuppression was observed, including decreases in
Administered i.v. RBCs, Hb, Hct, and WBCs. Findings were prominent in
the high dose groups.
per sex per Gross necropsy revealed atrophy of the lymphoid tissues,
group; 3 mg/kg and spleen, lymph nodes, gut associated with lymphoid
8 mg/kg groups tissue, and cellularity of the lamina propria of the
also included an intestine.
additional 5 Histopathology exams concluded that there were a
recovery animals number of organs with treatment effects; however, only
per sex the bone marrow, lymphoid organs, digestive tract, and
injection site had lesions that were considered direct test
article findin s.
Dog/ 4 days 5 mg/kg (single MTD << 5 mg/kg Acute toxicity was observed with a
single 5 mg/kg dose
Beagle/ dose) of RTA 744 in the canine model. At this dose level overt
Female clinical signs of gastrointestinal toxicity were observed
I animal within 3 days of treatment, with mortality observed on
study day 4. A single dose of 5 mg/kg exceeded the
MTD.
Dog/ 46 days 0.2 mg/kg daily x 3 MTD =.5 mg/kg RTA 744 was escalated in the
same dog every 14-21
Beagle/ 0.3 mg/kg daily x 3 qdx3 days, from .2 to .3 and .5 mg/kg/day for 3
consecutive
Female 0.5 mg/kg daily x 3 days. Doses of 0.2 and 0.3 mg/kg daily x 3 produced
no
adverse clinical signs, but white blood cell and platelet
Administered i.v. counts decreased, with nadir at 8-10 days before
rebounding 4 to 6 days later.
I animal Following the third injection of .5 mg/kg x 3 days, mild
gastrointestinal toxicity was observed on day 5 and signs
of morbidity were observed on day 9. WBC and platelets
were profoundly depressed, and the animal was
euthanized.
Gross and microscopic evaluatiosn revealed a lack of
adverse findings, including normal serosal surface of all
segments of the gut; normal mucosal surface of most
areas of the gut, with linear hemorrhages in the large
intestine. Heart, lungs, spleen, kidneys, and bone
marrow all appeared normal.
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Species/
Strain/ Duration
Gender ofStudy Dose/Schedule Results Notewortity rindin s
Dog/ 17 days 0.25 ing/kg daily x MTD > 0.25 mg/kg RTA 744 was administered
daily for 3 consecutive days,
Beagle/ 3 MTD < 0.75 mg/kg followed by an 11 day rest period. After this rest
period,
Male & 0.75 ing/kg daily x qdx3 RTA 744 was administered for 3 more
consecutive days.
Female 3 All animals in the.75 and 1.25 mg/kg dose groups were
1.25 ing/kg daily x found dead or euthanized on Day 5. Clinical
3 observations ineluded inucoid stools, vomitus, decreased
activity, labored breatiiing, cool to touch, and
Administered i.v dehydration.
RTA 744 was well tolerated at .25 mg/kg/day, with the
1 per sex only remarlcable signs being soft stools and decreases in
total and some differential leukocyte counts.
Dog/ 28 days 0 mg/kg daily x 3 MTD = 0.33 qdx3 No deaths occurred at doses of
.25 mg/kg or.33 mg/kg.
Beagle/ 0.25 mg/kg daily x Clinical observations in these groups were limited
to
Male & 3 soft/mucoid stools.
Female 0.33 mg/kg daily x Three-fourths (9/12) of animals at the high dose
died or
3 were euthanized, including all females. Clinical
0.50 mg/kg daily x observations included decreased activity, salivation, no
3 food consumed, breathing abnormalities, abnormally
colored mucoid stools, dehydration, thin appearance, and
Administered i.v. vomitus.
Animals receiving RTA 744 exhibited
4-6 per sex per myelosuppression, including decreases in RBCs, Hb,
group (some Hct, WBCs, and platelets. All findings were prominent
animals were at the high dose.
assigned to a 14- Major gross findings included small tliymus, dark red
day recovery lungs, dark red mediastinal lymph node, pale small/ large
group) intestine, small spleen, linear striations on the colon,
cysts on the pituitary, dark red areas of the cecum, dark
red/mottled kidneys and reddened duodenum.
Test article related histopathology findings (including
hypocellularity of the bone marrow, necrosis and
ulceration in the intestines and consolidation in the lungs)
were largely reversible within the 14-day recovery
period.
EXAMPLE 7
Myocardial Toxicity Study
Due to the relationship between members of the anthracycline class and cardiac
toxicity, a myocardial toxicity study of WP744 with doxorubicin serving as a
positive control
was perfomed. Fifteen mice (CDl, female) per group were treated with vehicle,
doxorubicin
(1, 2, or 4 mg/kg), or WP744 (1, 2, or 4 mg/kg). All treatments were
administered as 2 doses
per week for 2 weeks, followed by a 2 week observation period, then 2 doses
additional per
week for 3 weeks, followed by a 4 week observation period prior to sacrifice
and necropsy.
Cardiotoxic effects of treatment were evaluated for severity and extent
according to
criteria established by Bertazzoli for quantifying the cardiotoxicity of
doxorubicin (Bertazzoli
et al., Cancer Treat Rep. 1979 Nov-Dec;63(11-12):1877-83). In this system, the
Bertazzoli
score is the product of the severity and extent scores (Table 9), allowing
comparison across
groups.
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Table 9 Calculation of Bertazzoli Score
Severity of Toxicity x Extent of Toxicity
I Sarcoplasmic microvacuolization and/or 0 No lesions
inclusions and interstitial cellular 0.5 < 10 single altered myocytes in the
whole
edema, heart section
2 Same as 1+ sarcoplasmic 1 Scattered single altered myocytes
macrovacuolizations or atrophia, 2 Scattered small groups of altered myocytes
necrosis, fibrosis, endocardial lesions 3 Widely spread small groups of
altered
and thrombi. myocytes
4 Confluent groups of altered myocytes
Most cells damaged
In this study, no animals died or had significant gross lesions upon necropsy.
Cardiotoxicity findings are summarized in Table 10. From these results, it is
inferred that
5 cumulative doses of WP744 produced less cardiotoxicity than equivalent doses
of
doxorubicin.
Table 10. Cardiotoxicity Assessment Summary - Bertazzoli Scores
Treatment Group Incidence of Sum of Mean Score Group Mean Sum of Other
Lesions (%)' Score2 Lesion Mice3 Score Lesions4
Controls 9 of 15 60%) 29 29/9 = 3.2 29/15 = 1.9 0
Doxorubicin
1 g/kg 13 of 15 87%) 41 41/13=3.2 41/15 = 2.7 10
2 mg/kg 15 of 15 100% 53.5 53.5/15 = 3.6 53.5/15 = 3.6 12
4 m/k 14 of 14 (100 %) 53 53/14 = 3.8 53/14 = 3.8 47
WP744
1 m/k 13o f 15 87% 42.5 42.5/13 = 3.3 42.5/15 = 2.8 7
2m /k 13of15 87% 35.5 35.5/13=2.7 35.5/15=2.4 2
4m /k 13of15 87% 30.5 30.5/13=2.3 30.5/15=2.0 6
incidence is based on the number of mice with lesions / number in group
2 score = product of the severity scores (1-2) and extent scores (0-5)
3 scores of mice with lesions / number of mice with lesions
4 Other lesion grades defined on examination as grossly evident degeneration
or necrosis,
scores from 1+ to 5+
EXAMPLE 8
Comparitive Toxicology of Doxorubicin, Epirubicin, and WP744
Based upon data from preclinical studies conducted with WP744, this compound
has a
similar spectrum of toxicity compared to other members, of the anthracycline
class, most
notably doxorubicin and epirubicin. A comparison of toxicology data for RTA
744 and
Summary Basis of Approval docuinentation for Adriamycin (doxorubicin) and
Ellence
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(epirubicin) indicates that WP744 has a preclinical toxicology profile that is
quite similar to
these other agents, with the exception being increased tolerability in
rodents.
The single dose LD50 for WP744 is higher than that for doxorubicin and
epirubicin in
both mice and rats (56 vs. 15.9 and 23 mg/kg in mice and 15-20 vs. 8.7 and 14-
15 in rats). In
dogs, the LD50 is closer ainong the agents, witli WP744 having the lowest
value (1.5-2.25 vs.
2.6 and 2). All data expressed in mg/kg. Schedule for all studies was a single
bolus injection
except for the WP744 dog study, in which drug was administered on a daily x 3
schedule.
EXAMPLE 9
Phase 1 Dose-Escalation Study
A Phase 1 dose-escalation study (Protocol RTA744-C-0401) is currently underway
to
evaluate the tolerability and antitumor activity of WP744 Injection given
intravenously for
three consecutive days of a three-week cycle in adult patients with recurrent
or refractory
primary brain tumors (glioblastoma multiforme, anaplastic astrocytoma,
anaplastic
oligodendroglioma, anaplastic mixed oligo-astrocytoma, or gliosarcoma). This
study is
designed to determine the maximum tolerated dose (MTD) and the dose-limiting
toxicities of
WP744 when administered in this patient population. Additionally, the study is
assessing the
safety, tolerability, and pharmacokinetic profile of WP744. MRI imaging at
baseline and
after even-numbered cycles is also being conducted to provide a preliminary
indicator of
activity.
Four patients have been treated in this trial at two dose levels, for a total
of 11 cycles.
Patients have received up to four cycles of therapy.
EXAMPLE 10
Human Treatment with C-3' or C-4' Substituted Anthracycline Compounds in
Combination With Alkylating Agents
This example describes a protocol to facilitate the treatment of cancer using
C-3' or C-
4' substituted anthracycline compounds (e.g. WP744) in combination with
alkylating agents
(e.g., temozolomide). For illustrative purposes only, WP744 will be used as
the C-4'
substituted anthracycline compound and temozolomide as the alkylating agent.
However, it is
contemplated that all substituted anthracycline coinpounds and alkylating
agents described in
this specification can be used.
A cancer patient presenting, for example, an MDR cancer is treated using the
following protocol. Patients may, but need not, have received previous chemo-
radio- or gene
therapeutic treatments. Optimally, the patient exhibits adequate bone marrow
function (e.g.,
peripheral absolute granulocyte count of > 2,000/mm3 and platelet count of
100, 000/mm3,
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adequate liver function (bilirubin 1.5mg/dl) and adequate renal function
(creatinine
1.5mg/dl)).
Exemplaiy Protocol for the Treatrnent of Multi-Drug Resistant Cancer:
Coinposition(s) of the present invention can be administered orally or
parenterally in dosage
unit fonnulations containing standard, well lcnown, non-toxic physiologically
acceptable
carriers, adjuvants, and vehicles as desired. The term parenteral encompasses
subcutaneous
injections, intravenous, intramuscular, intra-arterial injection, or infusion
techniques. WP744
and temozolomide can be delivered to the patient before, after, or
concurrently with the other
anti-cancer agents or other cancer therapies.
A non-liiniting treatinent course can include about six doses of each of WP744
and
temozolomide delivered over a 7- to 21-day period. Upon election by the
clinician, the
regiinen may be continued six doses every three weeks or on a less frequent
(monthly,
bimonthly, quarterly etc.) basis. Of course, these are only exemplary times
for treatment, and
the skilled practitioner will readily recognize that many other time-courses
are possible.
A major challenge in clinical oncology is that many cancers are multi-drug
resistant.
One goal has been to find ways to improve the efficacy of chemotherapy. In the
context of
the present invention, the combination of WP744 and temozolomide have a
surprising
cytotoxicity against cancers. To kill MDR cancer cells using the methods and
compositions
described in the present invention, one will generally contact a target cell
with WP744 and
temozolomide in amounts effective to kill or iiihibit the proliferation of the
cell.
In certain embodiments, it is contemplated that one would contact the cell
with
agent(s) of the present invention about every 6 hours to about every one week.
In some
situations, however, it may be desirable to extend the time period for
treatment significantly
where several days (2, 3, 4, 5, 6, 7 or more) to several weeks (1, 2, 3, 4, 5,
6, 7, or more) lapse
between respective administrations. Regional delivery of WP744 and
temozolomide is an
efficient method for delivering a therapeutically effective dose to counteract
the clinical
disease. Likewise, the chemotherapy may be directed to a particular affected
region.
Alternatively, systeinic delivery of active agents may be appropriate.
The therapeutic composition of the present invention can be administered to
the
patient directly at the site of the tumor. This is in essence a topical
treatment of the surface of
the cancer. The volume of the composition should usually be sufficient to
ensure that the
tumor is contacted by WP744 and temozolomide. In one embodinient,
administration simply
entails injection of therapeutic composition(s) into the tumor. In another
embodiment, a
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catheter is inserted into the site of the tumor, and the cavity may be
continuously perfused for
a desired period of time.
Clinical responses may be defined by acceptable measure. For example, a
complete
response may be defined by the disappearance of all measurable disease for at
least a month,
whereas a partial response may be defined by a 50% or greater reduction of the
suin of the
products of perpendicular diaineters of all evaluable tumor nodules or at
least one month with
no tumor sites showing enlargeinent. Similarly, a mixed response may be
defined by a
reduction of the product of perpendicular diameters of all measurable lesions
by 50% or
greater, with progression in one or more sites.
Of course, the above-described treatment regimes may be altered in accordance
with
the knowledge gained from clinical trials such as those described above. Those
of skill in the
art are able to take the infomlation disclosed in this specification and
optimize treatment
regimes based on the clinical trials described in the specification.
EXAMPLE 11
Human Treatment with C-3' or C-4' Substituted Anthracycline Compounds in
Combination With Allcylating Agents
This example explains the development of human treatment protocols using C-3'
or C-
4' substituted anthracycline compounds (e.g. WP744) in combination with
alkylating agents
(e.g., temozolomide). For illustrative purposes only, WP744 will be used as
the C-4'
substituted anthracycline compound and temozolomide as the alkylating agent.
However, it is
contemplated that all substituted anthracycline compounds and alkylating
agents described in
this specification can be used.
These compounds are of use in the clinical treatment of various MDR cancers in
which transforined or cancerous cells play a role. Such treatment is a
particularly useful tool
in anti-tumor therapy, for example, in treating patients with brain, ovarian,
breast and lung
cancers that are resistant to conventional chemotherapeutic regimens. The
various elements
of conducting a clinical trial, including patient treatment and monitoring, is
known to those of
skill in the art in light of the present disclosure. The following information
is being presented
as a general guideline for use in establishing substituted anthracyclines
drugs made by the use
of this invention, in clinical trials.
Patients with human brain cancer, metastatic breast and/or epithelial ovarian
carcinoma, colon cancer leukemia, or sarcoma are chosen for clinical study.
Measurable
disease is not required, however the patient must have easily accessible
pleural effusion and/or
ascites. Further the patients must carry tumors that express MDR phenotype. In
an exemplary
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clinical protocol, patients may undergo placement of a Tenckhoff catheter, or
other suitable
device, in the pleural or peritoneal cavity and undergo serial satnpling of
pleural/peritoneal
effusion. Typically, one will wish to determine the absence of lctiown
loculation of the pleural or
peritoneal cavity, creatinine levels that are below 2 mg/dl, and bilirubin
levels that are below 2
mg/dl. The patient should exhibit a norinal coagulation profile.
In regard to WP744 and temozolomide administration, a Tenckhoff catheter, or
alternative device, may be placed in the pleural cavity or in the peritoneal
cavity, unless such a
device is already in place from prior surgery. A sainple of pleural or
peritoneal fluid can be
obtained, so that baseline cellularity, cytology, LDH, and appropriate markers
in the fluid (CEA,
CA15-3, CA 125, p185) and in the cells (E1A, p185) may be assessed and
recorded.
In the same procedure, WP744 and teinozolomide can be administered. The
administration may be in the pleural/peritoneal cavity, directly into the
tuinor, or in a systemic
manner. The starting dose may be 0.5mg/kg body weight. Three patients may be
treated at each
dose level in the absence of grade > 3 toxicity. Dose escalation may be done
by 100%
increments (0.5mg, lmg, 2mg, 4mg) until drug related Grade II toxicity is
detected. Thereafter,
dose escalation may proceed by 25% increments. The administered dose may be
fractionated
equally into two infusions, separated by 6 hours if the combined endotoxin
levels determined for
the lot of bisantliracycline exceed 5EU/kg for any given patient.
WP744 and temozolomide can be administered over a short infusion time or at a
steady rate of infusion over a 7- to 21-day period. The infusion given at any
dose level is
dependent upon the toxicity achieved after each. Hence, if Grade II toxicity
was reached after
any single infusion, or at a particular period of time for a steady rate
infusion, further doses
should be withheld or the steady rate infusion stopped unless toxicity
improves. Increasing
doses of WP744 and/or temozolomide in combination with other an anti-cancer
drugs or
therapies can be administered to groups of patients until approximately 60% of
patients show
unacceptable Grade III or IV toxicity in any category. Doses that are 2/3 of
this value could
be defined as the safe dose.
Physical examination, tumor measurements, and laboratory tests can be
performed
before treatment and at intervals of about 3-4 weeks later. Laboratory studies
can include
CBC, differential and platelet count, urinalysis, SMA-12-100 (liver and renal
function tests),
coagulation profile, and any other appropriate chemistry studies to determine
the extent of
disease, or determine the cause of existing symptoms. Also appropriate
biological markers in
serum can be monitored, (e.g., CEA, CA 15-3, p185 for breast cancer, and CA
125, p185 for
ovarian cancer).
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To monitor disease course and evaluate the anti-tuinor responses, it is
contemplated
that the patients can be exainined for appropriate tumor marlcers every 4
weeks, if initially
abnormal, with twice weeldy CBC, differential and platelet count for the 4
weelcs. Theii, if no
inyelosuppression has been observed, weekly. If any patient has prolonged
inyelosuppression, a
bone manow exainination is advised to rule out the possibility of ttunor
invasion of the maiTow
as the cause of pancytopenia. Coagulation profile shall be obtained every 4
weeks. An
SMA-12-100 shall be performed weeldy. Pleural/peritoneal effusion may be
sampled 72 hours
after the first dose, weekly thereafter for the first two courses, then every
4 weelcs until
progression or off study. Cellularity, cytology, LDH, and appropriate markers
in the fluid (CEA,
CA15-3, CA 125, p185) and in the cells (p185) may be assessed. An example of
an evaluation
profile is shown in Table 11. When measurable disease is present, tumor
measurements are to be
recorded every 4 weeks. Appropriate radiological studies should be repeated
every 8 weeks to
evaluate tumor response. Spirometry and DLCO may be repeated 4 and 8 weeks
after initiation
of therapy and at the tiine study participation ends. A urinalysis may be
performed every 4
weeks.
Table 11. Evaluations Before and During Therapy
EVALUATIONS PRE- TWICE WEEKLY EVERY 4 EVERY 8
STUDY WEEKLY WEEKS WEEKS
History X X
Physical X p X
Tumor Measurements X X
CBC X X' X
Differential X X' X
Platelet Count X Xl X
SMA12-100 (SGPT, X X
Alkaline Phosphatase,
Bilirubin, Alb/Total Protein)
Coagulation Profile X X
Serum Tumor markers X X3
(CEA, CA15-3, CA-125,
Her-2/neu)
Urinalysis X X
X-rays:
chest X X4
others X X
PleuraUPeritoneal Fluids: X X5 X
(cellularity, cytology, LDH,
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EVALUATIONS PRE- TWICE WEEICLY EVERY 4 EVERY 8
STUDY WEEKLY WEEKS WEEKS
tumor markers, E1A, IIER-
2/neu)
Spirometry and DLCO X XG X6
1 For the first 4 weeks, then weeldy, if no myelosuppression is observed.
2 As indicated by the patient's condition.
3 Repeated every 4 weeks if initially abnormal.
4 For patients with pleural effusion, chest X-rays may be performed at 72
hours after first dose, then prior to
each treatment administration.
5 Fluids may be assessed 72 hours after the first dose, weeldy for the first
two courses and then every 4 weeks
thereafter.
6 Four and eight weeks after initiation of therapy.
All of the compositions and/or methods disclosed and claimed in this
specification can
be made and executed witllout undue experimentation in light of the present
disclosure.
While the compositions and methods of this invention have been described in
terms of
preferred embodiments, it will be apparent to those of skill in the art that
variations may be
applied to the compositions and/or methods and in the steps or in the sequence
of steps of the
method described herein without departing from the concept, spirit and scope
of the invention.
More specifically, it will be apparent that certain agents which are both
chemically and
physiologically related may be substituted for the agents described herein
while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to
those skilled in the art are deemed to be within the spirit, scope and concept
of the invention
as defined by the appended claims.
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REFERENCES
The following references, to the extent that they provide exemplary procedural
or
other details suppleinentary to those set forth herein, are specifically
incoiporated herein by
reference.
U.S. Patent No. 6,673,907
Bertazzoli et al., Cancer Treat Reu. 1979 Nov-Dec; 63(11-12):1877-83.
Danesi R, Fogli S, Gennari A, et al. Pharinacokinetic-pharmacodynamic
relationships of the
anthracycline anticancer drugs. Clin. Pharmacokinet. 41:431-44, 2002.
Faderl S, Estrov Z, Kantarjian HM, et al. WP744, a novel anthracycline with
enhanced
proapoptotic and antileukemic activity. Anticancer Res. 21:3777-84, 2001.
Gustafson DL, Rastatter JC, Colombo T, et al. Doxorubicin pharmacokinetics:
macromolecule binding, metabolism, and excretion in the context of a
physiologic
model. J Pharm. Sci. 91:1488-501, 2002.
