Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF CANCER PATIENTS EXHIBITING ACTIVATION
OF THE P-GLYCOPROTEIN EFFLUX PUMP MECHANISM
RELATED APPLICATION
This application claims priority to U.S. Provisional Application No.
60/696,913
filed July 6, 2005, U.S. Application No. 11/418,399 filed May 3, 2006, U.S.
Application
No. 11/417,984 filed May 3, 2006, U.S. Application No. 11/418,323 filed May 3,
2006,
and to U.S. Provisional Application No. 60/726,827 filed October 14, 2005,
which are
expressly incorporated by reference herein in their entirety, and which are
hereby made a
part of this specification.
FIELD OF THE INVENTION
The present invention relates to a method of determining P-glycoprotein
expression and/or function for a patient with solid tumors, leukemias, and
other
malignancies. The invention also relates to using a P-glycoprotein expression
and/or
function diagnostic in conjunction with methods for treating solid tumors,
leukemias, and
other malignancies with a chemotherapeutic agent in combination with
zosuquidar. The
methods are particularly effective in treating acute myelogenous leukemia,
metastatic
breast cancer, and other cancers expressing P-glycoprotein, wherein the P-
glycoprotein
expression and/or function is used to select a treatment option for the
patient.
BACKGROUND OF THE INVENTION
The field of oncology is in the midst of a major evolution. In the past, the
treatment of cancer has been dominated by empiric, "one-size-fits-all"
treatments based
on types and stages of tumors. Toxic chemotherapy drugs have dominated the
treatment
landscape despite a very low cure rate, particularly against the most common
cancers and
those with known metastatic disease.
Now, treatments in development are targeted against specific proteins. Such
targeting is based on a more robust knowledge of cancer mechanisms, which
often crosses
over many tumor types. These treatments are designed to work in defined
subsets of
patients, typically based on expression and function of the target protein
rather than the
type of tumor, and often in combination with standard chemotherapies. Advances
in the
molecular analysis of cancers will enable the identification of such susbsets
of patients
and the coupling of targeted therapeutics to novel diagnostic approaches.
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The future of oncology lies in defining the disease in molecular terms (i.e.,
genetics, genomics, proteomics) and tailoring therapies according to
individual tumor and
normal cell properties. This new paradigm will predetermine likely responders,
assess
responses earlier, and adjust treatment based on continued molecular analyses
of tumors.
Drug resistance is one of the most difficult problems that must be overcome in
order to achieve successful treatment of human tuinors with chemotherapy.
Clinically,
drug resistance, a characteristic of intrinsically resistant tumors (for
example, colon, renal,
and pancreas), may be evident at the onset of therapy. Alternatively, acquired
drug
resistance results when tumors initially respond to therapy but become
refractory to
subsequent treatments. Once a tumor has acquired resistance to a specific
chemotherapeutic agent, it is common to observe collateral resistance to other
structurally
similar agents. The cellular mechanisms of drug resistance include apoptosis,
drug
uptake, DNA repair, altered drug targets, drug sequestration, detoxification,
and efflux
pumps (see, e.g., Dalton W.S. Semin. Oncol. 20:60, 1993).
Multidrug resistance (MDR), the ability of cancer cells to become resistant to
the
agent(s) actively used for therapy as well as other drugs that are
structurally and
functionally unrelated, is a particularly insidious form of drug resistance.
This form of
drug resistance is discussed in greater detail in Kuzmich et al.,
"Detoxification
Mechanisms and Tumor Cell Resistance to Anticancer Drugs," particularly
section VII
"The Multidrug-Resistant Phenqtype (MDR)," Medical Research Reviews, Vol. 11,
No.
2, 185-217, particularly 208-213 (1991); and in Georges et al., "Multidrug
Resistance and
Chemosensitization: Therapeutic Implications for Cancer Chemotherapy,"
Advances in
Pharmacology, Vol. 21, 185-220 (1990).
Although MDR may be caused by a variety of factors, one of the most prevalent
forms of MDR is the type associated with overexpression of P-glycoprotein (P-
gp). P-gp
is a member of a superfamily of membrane proteins, termed adenosine
triphosphate
(ATP)-binding cassette (ABC) proteins, which behave as ATP-dependent
transporters
and/or ion channels for a wide variety of hydrophobic substrates. P-gp is a
multiple
transmembrane-spanning glycoprotein. Transfection experiments with the P-gp
gene
(MDR1, or ABCBI) have conferred MDR to drug-sensitive tumor cells by providing
an
energy-dependent efflux pump that lowers the intracellular concentration of
the cytotoxic
agent, thereby allowing survival of the cell.
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P-gp is expressed in normal biliary canaliculi of the liver, the adrenal
cortex and
proximal tubules of the kidney, and intestinal epithelia including the
columnar cells of the
large and small intestines; capillary endothelial cells of brain, testis, and
placenta; and in
the hematopoietic stem cells of bone marrow. It possesses excretory,
protective, and
barrier functions. P-gp is constitutively expressed or selected in many human
cancers,
and confers resistance to therapeutic agents including anthracyclines (e.g.,
doxorubicin,
daunorubicin, epirubicin, idarubicin, mitoxantrone), vincas (e.g.,
vincristine, vinblastine,
vinorelbine, vindesine), Topoisomerase-II inhibitors (e.g, etoposide,
teniposide), taxanes
(e.g., paclitaxel, docetaxel), and others (e.g., Gleevec, Mylotarg,
dactinomycin,
mithramycin).
The relative promiscuity of drug transport by P-gp and other MDR-associated
transporters inspired a wide search for compounds that would not be cytotoxic
themselves
but would inhibit MDR transport. The initial demonstration of verapamil as a P-
gp
inhibitor was followed by many additional compounds reported to inhibit drug
transport
and thus sensitize MDR cells to chemotherapeutic drugs. Variously called
chemosensitizers, MDR reversal agents, modulators, or converters, these 'first
generation'
MDR drugs included compounds of diverse structure and function such as calcium
channel blockers (e.g., verapamil), immunosuppressants (e.g., cyclosporin A),
antibiotics
(e.g., erythromycin), antimalarials (e.g., quinine), and others (e.g.,
biricodar, tariquidar,
valspodar).
First generation MDR drugs were not specifically developed for inhibiting MDR.
They often had other pharmacological activities, as well as a relatively low
affinity for
MDR transporters and thus were limited in application. For example, P-gp has a
low
affinity for verapamil, thus requiring cardiotoxic levels for full modulator
activity. In
spite of the fact that only low serum levels could be obtained in a Phase II
trial, 5 of 22
patients responded to a combination of verapamil and VAI) (vincristine,
doxorubicin, and
dexamethasone). Four of the responders had elevated P-gp expression and
function.
Thus, verapamil has demonstrated some clinical utility in overcoming drug
resistance.
Cyclosporin A alters the pharmacokinetics of coadministered cytotoxic agents,
resulting
in significantly increased exposure to the oncolytic, thus confounding the
interpretation of
clinical trials.
Further characterization of the P-gp pharmacophore led to the identification
of
'second generation' modulators based on the first generation but specifically
selected or
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designed to reduce the side effects of the latter by eliminating their non-MDR
pharmacological actions. Compounds such as the R-enantiomers of verapamil (R-
verapamil) and dexniguldipine did not fare any better as MDR drugs in clinical
studies,
most likely because their affinity towards P-gp still fell short of producing
significant
inhibition of MDR in vivo at tolerable doses.
A more promising second generation modulator with a higher affinity towards P-
gp was valspodar, a non-immunosuppressive cyclosporin D derivative. While
early trials
were encouraging, further work revealed significant pharmacokinetic
interactions with
several anticancer drugs. Although discontinued by Novartis, valspodar was
studied in a
Phase III study in elderly patients with acute myelogenous leulcemia.
Enrollment in the
valspodar arm was halted due to excessive early mortality, most likely due to
the PK
interactions. Although the number of patients was limited, patients in the
control arm
wllose pretreatment cells exhibited valspodar-modulated dye efflux in vitro (n
= 22) had
worse outcomes than those without efflux (n = 11) (complete remission,
nonresponse, and
death rates of 41%, 41%, and 18%, compared with 91%, 9%, and 0%; P = 0.03),
but with
valspodar outcomes were nearly identical (Baer 2002). Moreover, for patients
with
valspodar-modulated efflux, median disease-free survival was 5 months in the
control arm
and 14 months with valspodar (P = 0.07).
A second generation MDR modulator with activity against both P-gp and MRP 1
(another ABC transporter associated with multidrug resistance) was biricodar.
Vertex
studied the agent in multiple Phase II studies of soft tissue sarcomas,
ovarian cancer,
small cell lung cancer, and others. However, biricodar and valspodar are both
substrates
for the P450 isoenzyme 3A4. Competition between cytotoxic agents and the P-gp
inhibitors for cytochrome P450 3A4 resulted in unpredictable PK interactions
and resulted
in increased serum concentrations of cytoxics and, therefore, greater toxicity
to the
patient. A common response of clinical researchers has been to reduce the dose
of the
cytotoxic agents. However, the PK interactions are unpredictable and cannot be
determined in advance. As a result, cytotoxic serum levels were either too
high resulting
in excessive toxicity or too low resulting in decreased efficacy. In addition
to inhibiting
P-gp, many of the second generation modulators function as substrates for
other
transporters, particularly the ABC family, inhibition of which could lessen
the ability of
normal, healthy cells to protect themselves from the cytotoxic agents.
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SUMMARY OF THE INVENTION
In spite of the methodological possibilities for detecting expression of MDR1,
the
use of P-gp expression and functional tests to predict response to therapy has
not been
very successful. Specifically, while there have been indications of clinical
utility for such
a test, the results have not been sufficiently strong to warrant routine
testing. What has
been lacking is a statistical algorithm relating response potential to
chemotherapy on a
patient-specific basis with P-gp function to the modulatory effects of
zosuquidar, the
iinmunophenotype, and cytogenetics of the cancer cells, and clinical data such
as patient
age.
Zosuquidar selectively inhibits MDRl activity, allowing for the accumulation
of
sufficient chemotherapeutic agent to allow 'for effective therapy and tumor
cell killing.
MDR1 may be present in the tumor cells at the time of diagnosis or it may be
acquired
during treatment or remission of the cancer. It is desirable to identify
patients who would
benefit most from zosuquidar therapy to target that patient population, i.e.,
MDRl-
positive. A highly sensitive and reproducible diagnostic test for the
functional expression
of MDR1 is required to accomplish this goal.
Testing methodology is provided that improves the efficacy rates for any P-gp
substrate in a patient population with demonstrated pump activation, resulting
in
enhanced cure rates, cancer free survival rates, and overall survival rates
for oncolytic
drugs.
Accordingly, in a first aspect a method of treating a cancer is provided, the
method comprising the steps of determining P-glycoprotein expression or P-
glycoprotein
function in the cancer cells; selecting a treatment for the patient, based on
the P-
glycoprotein expression; and administering the treatment, whereby the cancer
is treated.
In an embodiment of the first aspect, P-glycoprotein expression is determined
by
an antibody assay, and the step of selecting the treatment and administering
the treatment
comprises administering a P-glycoprotein efflux pump inhibitor in combination
with,a
chemotherapeutic agent that is a substrate for P-glycoprotein efflux when
positive P-
glycoprotein expression is observed in at least about 10% of the cells tested
in the
antibody assay. Positive P-glycoprotein expression can be observed in from
about 10% to
about 25% of the cells tested in the antibody assay.
In an embodiment of the first aspect, P-glycoprotein expression is deterinined
by
an antibody assay, and the step of selecting the treatment and administering
the treatment
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comprises administering a chemotherapeutic agent that is a substrate for P-
glycoprotein
efflux in the absence of a P-glycoprotein efflux pump inhibitor when positive
P-
glycoprotein expression is observed for less than about 10% of the cells
tested in the
antibody assay.
In an embodiment of the first aspect, P-glycoprotein function is determined by
a P-
glycoprotein dye accumulation assay, and the step of selecting the treatment
and
administering the treatment comprises administering a P-glycoprotein efflux
pump
inhibitor in combination with a chemotherapeutic agent that is a substrate for
P-
glycoprotein efflux when, in the P-glycoprotein dye accumulation assay, a
ratio of dye
taken up by cells in the presence of the P-glycoprotein efflux pump inhibitor
to dye taken
up by cells cultured in the absence of the P-glycoprotein efflux pump
inhibitor is at least
about 1:1.2, e.g., from about 1:1.2 to about 1:1.5.
In an erribodiment of the first aspect, P-glycoprotein function is determined
by a P-
glycoprotein dye accumulation assay, and the step of selecting the treatment
and
administering the treatment comprises administering a chemotherapeutic agent
that is a
substrate for P-glycoprotein efflux in the absence of a P-glycoprotein efflux
pump
inhibitor when, in the P-glycoprotein dye accumulation assay, a ratio of dye
taken up by
cells in the presence of the P-glycoprotein efflux pump inhibitor to dye taken
up by cells
cultured in the absence of the P-glycoprotein efflux pump inhibitor is less
than about
1:1.2.
In an embodiment of the first aspect, P-glycoprotein function is determined by
a P-
glycoprotein dye efflux assay, and the step of selecting the treatment and
administering
the treatment comprises administering a P-glycoprotein efflux pump inhibitor
in
combination with a chemotherapeutic agent that is a substrate for P-
glycoprotein efflux
wlien, in the P-glycoprotein dye efflux assay, an amount of dye eliminated
from the cells
in the presence of the P-glycoprotein efflux pump inhibitor divided by an
amount of dye
eliminated in the absence of P-glycoprotein efflux pump inhibitor is at least
about 1:1.2,
e.g., from about 1:1.2 to about 1:1.5.
In an embodiment of the first aspect, P-glycoprotein function is determined by
a P-
glycoprotein dye efflux assay, and the step of selecting the treatment and
administering
the treatment comprises administering a P-glycoprotein efflux pump inhibitor
in
combination with a chemotherapeutic agent that is a substrate for P-
glycoprotein efflux
when, in the P-glycoprotein dye efflux assay, an amount of dye eliminated from
the cells
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in an absence of the P-glycoprotein efflux pump inhibitor is at least about
30% higher
than that contained in baseline control cells, and cells incubated in a
presence of the P-
glycoprotein efflux pump inhibitor can have dye levels at least about 30%
higher than
cells cultured in an absence of the P-glycoprotein efflux pump inhibitor. An
amount of
dye eliminated from the cells in an absence of the P-glycoprotein efflux pump
inhibitor
can be from about 30% to about 50% higher than that contained in baseline
control cells,
and cells incubated in a presence of the P-glycoprotein efflux pump inhibitor
can have dye
levels from about 30% to about 50% higher than cells cultured in an absence of
the P-
glycoprotein efflux pump inhibitor.
In an embodiment of the first aspect, P-glycoprotein function is determined by
a P-
glycoprotein dye efflux assay, and the step of selecting the treatment and
administering
the treatment comprises administering a chemotherapeutic agent that is a
substrate for P-
glycoprotein efflux in the absence of a P-glycoprotein efflux pump inhibitor
when, in the
P-glycoprotein dye efflux assay, an amount of dye eliminated from the cells in
the
presence of the P-glycoprotein efflux pump inhibitor divided by an amount of
dye
eliminated in the absence of P-glycoprotein efflux pump inhibitor is less than
about 1:1.2.
In an embodiment of the first aspect, P-glycoprotein function is determined by
a P-
glycoprotein dye efflux assay, and the step of selecting the treatment and
administering
the treatment comprises administering a chemotherapeutic agent that is a
substrate for P-
glycoprotein efflux in the absence of a P-glycoprotein efflux pump inhibitor
when, in the
P-glycoprotein dye efflux assay, an amount of dye eliminated from the cells in
an absence
of the P-glycoprotein efflux pump inhibitor is less than about 30% higher than
that
contained in baseline control cells, and cells incubated in a presence of the
P-glycoprotein
efflux pump inhibitor have dye levels less than about 30% higher than cells
cultured in an
absence of the P-glycoprotein efflux pump inhibitor.
In an embodiment of the first aspect, the P-glycoprotein efflux pump inhibitor
is
selected from the group consisting of zosuquidar, Tariquidar, or Tesmilifene.
In an embodiment of the first aspect, the cancer is acute myelogenous
leukemia.
In an embodiment of the first aspect, the cancer is a carcinoma, e.g., breast
cancer
or ovarian cancer.
In an embodiment of the first aspect, the cancer is a sarcoma.
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In an embodiment of the first aspect, the cancer is a hematologic malignancy,
e.g.,
acute lymphoblastic leukemia, chronic myeloid leukemia, plasma cell
dyscrasias,
lymphoma, or myelodysplasia.
In an embodiment of the first aspect, the chemotherapeutic agent is an
anthracycline, e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, or
mitoxantrone.
In an embodiment of the first aspect, the chemotherapeutic agent is a
Topoisomerase-II inhibitor, e.g., etoposide or teniposide.
In an embodiment of the first aspect, the chemotherapeutic agent is a vinca,
e.g.,
vincristine, vinblastine, vinorelbine, or vindesine.
In an embodiment of the first aspect, the chemotherapeutic agent is a taxane,
e.g.,
paclitaxel or docetaxel.
In an embodiment of the first aspect, the chemotherapeutic agent is selected
from
the group consisting of gleevec, dactinomycin, bisantrene, mitoxantrone,
actinomyocin D,
mithomycin C, mitramycin, methotrexate, adriamycin, mitomycin, mithramycin,
anthracene, or epipodophyllo-toxin.
In a second aspect a pharmaceutical kit is provided, the kit comprising at
least one
dose of a P-glycoprotein efflux pump inhibitor selected from the group
consisting of
zosuquidar, Tariquidar, and Tesmilifene; directions for conducting a
diagnostic for
determining P -glycoprotein expression or P-glycoprotein function associated
with the
cancer; and directions for administering the P-glycoprotein efflux pump
inhibitor and a
chemotherapeutic agent that is a substrate for P-glycoprotein efflux to the
patient to treat
the cancer when P-glycoprotein expression or P-glycoprotein function is
positive.
In a third aspect a method of treating a condition in a patient by
administering a
therapeutic agent that is a substrate for P-glycoprotein efflux is provided,
the method
comprising the steps of determining P-glycoprotein expression or P-
glycoprotein
function; and administering the therapeutic agent in combination with a P-
glycoprotein
efflux pump inhibitor when P-gp expression is positive.
In an embodiment of the third aspect, P-glycoprotein expression is determined
by
an antibody assay, and positive P-glycoprotein expression is observed in at
least about
10%, e.g., from about 10% to about 25%, of the cells tested in the antibody
assay.
In an embodiment of the third aspect, P-glycoprotein function is determined by
a
P-glycoprotein dye accumulation assay, and a ratio of dye taken up by cells in
the
presence of the P-glycoprotein efflux pump inhibitor to dye taken up by cells
cultured in
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the absence of the P-glycoprotein efflux pump inhibitor is at least about
1:1.2, e.g., from
about 1:1.2 to about 1:1.5.
In an embodiment of the third aspect, P-glycoprotein function is determined by
a
P-glycoprotein dye efflux assay, and an amount of dye eliminated from the
cells in the
presence of the P-glycoprotein efflux pump inhibitor divided by an amount of
dye
eliminated in the absence of P-glycoprotein efflux pump inhibitor is at least
about 1:1.2,
e.g., from about 1:1.2 to about 1:1.5.
In an embodiment of the third aspect, P-glycoprotein function is determined by
a
P-glycoprotein dye efflux assay, and an amount of dye eliminated from the
cells in an
absence of the P-glycoprotein efflux pump inhibitor is at least about 30%
higher than that
contained in baseline control cells, and cells incubated in a presence of the
P-glycoprotein
efflux pump inhibitor have dye levels at least about 30% higher than cells
cultured in an
absence of the P-glycoprotein efflux pump inhibitor.
In an embodiment of the third aspect, the P-glycoprotein efflux pump inhibitor
is
selected from the group consisting of zosuquidar, Tariquidar, or Tesmilifene.
In an embodiment of the third aspect, the therapeutic agent comprises an
immunosuppressant, e.g., cyclosporine, cyclosporine A, or tacrolimus.
In an embodiment of the third aspect, the therapeutic agent comprises a
steroid,
e.g., dexamethasone, hydrocortisone, corticosterone, triamcinolone,
aldosterone, or
methylprednisolone.
In an embodiment of the third aspect, the therapeutic agent comprises an
antiepileptic, e.g., phenytoin.
In an embodiment of the third aspect, the therapeutic agent comprises an
antidepressant, e.g., citalopram, thioperidone, trazodone, trimipramine,
amitriptyline, or
phenothiazines.
In an embodiment of the third aspect, the therapeutic agent comprises an
antipsychotic, e.g., fluphenazine, haloperidol, thioridazine, or trimipramine.
In an embodiment of the third aspect, the therapeutic agent comprises a
protease
inhibitor, e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, or
saquinavir.
In an embodiment of the third aspect, the therapeutic agent comprises a
calcium
blocker, e.g., bepridil, diltiazem, flunarizine, lomerizine, secoverine,
tamolarizine,
verapamil, nicardipine, prenylamine, or fendiline.
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In an embodiment of the third aspect, the therapeutic agent comprises a
cardiac
drug, e.g., digoxin, diltiazem, verapamil, or talinolol.
In a fourth aspect, a pharmaceutical kit is provided, the kit comprising at
least one
dose of a P-glycoprotein efflux pump inhibitor selected from the group
consisting of
zosuquidar, Tariquidar, and Tesmilifene; directions for conducting a
diagnostic for
determining a value for P-gp expression associated with a condition; and
directions for
administering the P-glycoprotein efflux pump inhibitor and a therapeutic agent
that is a
substrate for P-glycoprotein efflux to the patient to treat the condition when
P-
glycoprotein expression or P-glycoprotein function is pbsitive.
In a fifth aspect, a method of treating a cancer in a patient is provided, the
method
comprising the steps of conducting a P-glycoprotein efflux assay on the cancer
cells in the
presence of a P-glycoprotein efflux inhibitor, whereby a first value for P-
glycoprotein
function is obtained; conducting a P-glycoprotein efflux assay on the cancer
cells in the
absence of a P-glycoprotein efflux inhibitor, whereby a second value for P-
glycoprotein
function is obtained; comparing the first value and the second value for P-
glycoprotein
function, wherein the patient exhibits inhibitable P-glycoprotein efflux when
the first
value is greater than the second value; and administering to the patient
exhibiting
inhibitable P-glycoprotein efflux the P-glycoprotein efflux inhibitor and a
chemotherapeutic agent that is a substrate for P-gp efflux.
In an embodiment of the fifth aspect, the patient exhibits inhibitable P-
glycoprotein efflux when a ratio of the second value to the first value is
greater than or
equal to about 1:1.2.
In an embodiment of the fifth aspect, the patient exhibits inhibitable P-
glycoprotein efflux when a ratio of the second value to the first value is
from about 1:1.2
to about 1:1.5.
In an embQdiment of the fifth aspect, the patient exhibits inhibitable P-
glycoprotein efflux when the first value is at least about 30% higher than the
second
value.
In an embodiment of the fifth aspect, the patient exhibits inhibitable P-
glycoprotein efflux when the first value is from about 30% to about 50% higher
than the
second value.
In an embodiment of the fifth aspect, the P-glycoprotein efflux pump inhibitor
selected from the group consisting of zosuquidar, Tariquidar, and Tesmilifene.
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In an embodiment of the fifth aspect, the step of determining a value for P-gp
expression comprises conducting an assay, wherein the assay is selected based
on the
cancer.
In an embodiment of the fifth aspect, the cancer is a leukemia or a lymphoma,
and
wherein the step of determining a value for P-gp expression comprises
conducting a flow
cytometry assay.
In an embodiment of the fifth aspect, the cancer is a leukemia or a lymphoma,
and
wherein the step of determining a value for P-gp expression coinprises
conducting a
radiolabeled drug assay.
In an embodiment of the fifth aspect, the cancer is a leukemia or a lymphoma,
and
wherein the step of determining a value for P-gp expression
comprises.conducting a solid
phase immunoassay that measure a cell-associated drug with an anti-drug
antibody.
In an embodiment of the fifth aspect, the cancer is a solid tumor, and wherein
the
step of determining a value for P-gp expression comprises conducting an
immunocytochemistry assay.
In an embodiment of the fifth aspect, the cancer is a solid tumor, and wherein
the
step of determining a value for P-gp expression comprises conducting an
immunohistochemistry assay.
In an embodiment of the fifth aspect, the assay quantifies P-gp function.
In an embodiment of the fifth aspect, the cancer is acute myelogenous
leukemia.
In an embodiment of the fifth aspect, the cancer is a carcinoma, e.g., breast
cancer
or ovarian cancer.
In an embodiment of the fifth aspect, the cancer is a sarcoma.
In an embodiment of the fifth aspect, the cancer is a hematologic malignancy,
e.g.,
acute lymphoblastic leukemia, chronic myeloid leukemia, plasma cell
dyscrasias,
lymphoma, or myelodysplasia.
In an embodiment of the fifth aspect, the chemotherapeutic agent is an
anthracycline, e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, or
mitoxantrone.
In an embodiment of the fifth aspect, the chemotherapeutic agent is a
Topoisomerase-II inhibitor, e.g., etoposide or teniposide.
In an embodiment of the fifth aspect, the chemotherapeutic agent is a vinca,
e.g.,
vincristine, vinblastine, vinorelbine, or vindesine.
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In an embodiment of the fifth aspect, the chemotherapeutic agent is a taxane,
e.g.,
paclitaxel or docetaxel.
In an embodiment of the fifth aspect, the chemotherapeutic agent is selected
from
the group consisting of Gleevec, dactinomycin, mitomycin, mithramycin, and
Mylotarg.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 presents typical histograms of K562/R7 cells cultured in the absence
of
DiOC2, in the absence of zosuquidar, and in the presence of zosuquidar.
Figure 2 provides a comparison between accumulation and efflux assays with
leukemia cells.
Figure 3 provides a coinparison of efflux and accumulation assay data for
leukemia cells in the absence or presence of zosuquidar.
Figure 4 presents the efflux characteristics of K562/R7 cells loaded with 40
ng/ml
DiOC2 in the presence of 200 ng/ml zosuquidar for 60 minutes.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The following description and examples illustrate a preferred embodiment of
the
present invention in detail. Those of skill in the art will recognize that
there are numerous
variations and modifications of this invention that are encompassed by its
scope.
Accordingly, the description of a preferred embodiment should not be deemed to
limit the
scope of the present invention.
Cancer Targets
Many forms of cancer express P-gp, and thus can benefit from the
administration
of a P-gp efflux pump inhibitor when treated with a chemotherapeutic agent
that is a
substrate for P-gp efflux. For example, most solid tumors, lymphomas, bladder
cancer,
pancreatic cancer, ovarian cancer, liver cancer, myeloma, and sarcoma are all
cancers with
a P-gp expression of greater than 50%. Lymphocytic leukemia also has a P-gp
expression
of greater than 50%. The P-gp expression of breast cancers is about 30%. For
metastatic
breast cancer, 63% express P-gp. The methods and formulations of preferred
embodiments are particularly efficacious in the treatment of any malignancy
exhibiting
some degree of P-gp expression and/or function, or in patients who are P-gp
positive.
One form of cancer characterized by high rates of P-gp expression is acute
myelogenous leukemia. There are approximately 11,000 new cases of AML per year
in
the United States and 9,000 new cases in the five major EU countries. In
addition, the
World Health Organization defines advanced myelodysplastic syndrome (MDS) as
AML.
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There are approximately 4,000 cases of advanced MDS in the US and 3,000 cases
in the
five major EU countries. As a result, the target patient population for
zosuquidar is
approximately 15,000 patients in the U.S. and 12,000 in the major European
markets.
Adult AML presents greater treatment challenges when compared to pediatric
AML (age < 15 years). Due in part to a more resilient patient population and a
more
sensitive disease, the 5 year survival rates for pediatric AML is 50% (late
1990s). In
contrast, due in part to multiple co-morbid conditions and a more resistant
disease, the 5
year survival rates for adult AML are only 13% (late 1990s). The 5 year
survival rate for
patients over 65 is only 7%.
Standard induction therapy in the U.S. for newly diagnosed AML patients is
cytarabine with either idarubicin or daunorubicin (both P-gp substrates). In
one study,
71% of AML patients greater than 60 years of age expressed moderate to high
levels of P-
gp. The expression was associated with a reduction in the complete remission
(CR) rate.
The CR rate for P-gp negative AML patients was 67% compared to 34% for P-gp
positive
patients. This combination of high levels of P-gp expression with the nearly
universal use
of drugs that are P-gp substrates provides a ready opportunity for the
coadministration of
a P-gp inhibitor in patients with AML.
Approximately 75% of AML patients are over age 60, and 71% are P-gp positive.
The expression was associated with a reduction in the complete remission (CR)
rate. The
CR rate for P-gp negative AML patients was 67% compared to 34% for P-gp
positive
patients. Clinical outcomes in terms of patient survival rates are
significantly better for
patients that are P-gp negative than for those that are P-gp positive - a 50%
survival rate
at approximately 3-4 months for P-gp positive patients, versus a 50% survival
rate at
approximately 15 months for P-gp negative patients. See Campos, et al., Blood,
79:473-
476, 1992.
Approximately 75% of AML patients will eventually relapse and be candidates
for
additional treatment. Relapsed AML. patients typically require prolonged
hospitalization,
and their prognosis is generally poor. Of these relapsed patients,
approximately 80% are
P-gp positive.
Over 129,000 metastatic breast cancer patients are treated with chemotherapy
in
the United States and Europe annually. Of these patients, over 81,000 are
treated with a
P-gp substrate. 41% of breast cancers express P-gp. For metastatic breast
cancer, 63%
express P-gp.
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Zosuquidar
U.S. Pat. Nos. 5,643,909 and 5,654,304 disclose a series of 10,11-
methanobenzosuberane derivatives useful in enliancing the efficacy of existing
cancer
chemotherapeutics and for treating multidrug resistance. One such derivative
having
good activity, oral bioavailability, and stability, is zosuquidar, a compound
of formula
(2R)-anti-5-
3 -[4-(10,11-difluoromethanodibenzosuber-5-yl)piperazin-l-yl]-2-
hydroxypropoxy } quinoline.
HO N
H~
F
~~~l"IIN N H O
H~
Zosuquidar
Given the limitations of previous generations of MDR modulators, three
preclinical critical success factors were identified and met for zosuquidar:
1) it is a potent
inhibitor of P-glycoprotein; 2) it is selective for P-glycoprotein; and 3) no
pharmacokinetic interaction with co-administered chemotherapy is observed.
Zosuquidar is extremely potent in vitro (Ki = 59 nM) and is among the most
active
modulators of P-gp-associated resistance described to date. Zosuquidar has
also
demonstrated good in vivo activity in preclinical animal studies. In addition,
the
compound does not appear to be a substrate for P-gp efflux, resulting in a
relatively long
duration of reversal activity in resistant cells even after the modulator has
been
withdrawn.
Another significant attribute of zosuquidar as an MDR modulator is the minimal
pharmacokinetic (PK) interactions with several oncolytics tested in
preclinical models.
Such minimal PK interaction permits normal doses of oncolytics to be
administered and
also a more straightforward interpretation of the clinical results.
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Assays for Multidrug, Resistance
There are a variety of techniques to detect expression of MDR1. The detection
and quantitation of MDR1 protein is typically achieve using immunological
techniques.
For hematopoietic cells such as those from leukemia or lymphoma patients, the
techniques include flow cytometry and fixed cells on microscope slides. The
cells are
treated with antibodies specific for the MDR1 protein, such as the mouse ARK-
16
monoclonal antibody. Such antibodies can be directly labeled with fluorescent
probe, or
detected using subsequent reagents such as goat anti-mouse IgG-FITC. Flow
cytometry
allows for direct quantitative determinations of the full spectrum of MDR1
expression
using channel number or fluorescence intensity. Microscopic examination of the
slide
preparations can give qualitative results (-, +, ++, and the like) or, in
conjunction with an
image analyzer, quantitative evaluations typically expressed in pixels.
For solid tumors, such as breast cancer, typically immunocytochemistry (ICC)
or
immunohistochemistry (IHC) techniques are employed. Using, for example, frozen
sections or paraffin blocks, the detection techniques are the same as
described for fixed
leukemia cells on microscope slides. Expression of MDR1 can also be achieved
with the
measurement of specific mRNA levels. Cell slides can be processed, and levels
of
mRNA discerned using basic molecular biology techniques such as quantitative
fluorescent PCR. Alternatively, the cells of interest can be lysed, processed,
and
following PCR of the mRNA, the product can be detected and quantitated
following gel
electrophoresis. Anti-sense targeting of MDR1 mRNA is also possible, followed
by
standard techniques for quantitative determinations. Radio-labeled probes
followed by
autoradiography or other radiodetection techniques can also be used to get a
relative
estimate of MDR1 protein or mRNA expression. Thus, there exists a broad range
of
methods for the detection and quantitation of the spectrum of MDR1 expression
exhibited
by a patient population.
The relative expression of MDR1 is possible in vivo. MDR1-specific antibodies
labeled with any number of detectable markers, such as radioactive compounds
detectable
with positron emission tomography (PET), single-photon emission computed
tomography
(SPECT) or compounds detectable with magnetic resonance imaging (MRI) can be
used
to assess MDR1 expression in patients with cancer.
Of equal importance to MDRl protein and mRNA expression is the quantitation
of MDR1 function, i.e., functional expression. MDR1 functions as a cytoplasmic
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membrane pump, effluxing compounds such as drugs and toxins from the cytoplasm
to
the exterior of the cell. Compounds acted on by MDR1 are termed MDR1
substrates:
Detection of MDR1 function therefore involves detection of substrate efflux,
such as
drugs or, alternatively, detecting efflux of surrogate fluorescent dye markers
as DiOC2
(3,3'-diethyloxacarbocyanine iodide) or Rhodamine 123 (Rh123, or 2-(6-amino-3-
imino-
3H-xanthen-9-yl)benzoic acid, methyl ester). For single cell suspensions, such
as blood
or bone marrow from leukemia patients, the cells are exposed in tissue culture
to a
substrate for MDR1, such as the aforementioned dye markers, radiolabeled
drugs, or
drugs that can be quantitated by other means such as fluorescence. At
physiological
temperature (37 C) the net accumulation of the substrate over time, in the
presence or
absence of specific MDRl inhibitors, gives an indication of the MDRl
functional activity
exhibited by the cells. Alternatively, the single cell suspension can be
exposed to the
substrate, i.e., loaded with, and subsequent efflux of the substrate over time
monitored at
physiological teinperature in the presence or absence of specific MDR1
inhibitors.
PET, SPECT, and MRI techniques can also be used to assess MDR1 function in
cancer patients. Thus, small organic chemicals as well as metal complexes
which serve as
MDR1 substrates can be rendered as radionuclides or other markers which are
detectable
by the imaging technologies. Additionally, functional expression in solid
tumors can be
more efficiently ascertained by ICC/IHC techniques with prior labeling of the
tumor cells
while in the patient.
Diagnostic Testing for P-gp Expression and Efflux Pump Activity
Diagnostic testing methods for P-gp expression and efflux pump activity can be
used to prospectively stratify patients for treatment optimization in treating
malignancies
exhibiting P-gp expression or function, such as acute myelogenous leukemia,
most solid
tumors, lymphomas, bladder cancer, pancreatic cancer, ovarian cancer, liver
cancer,
myeloma, lymphocytic leukemia, and sarcoma.
In a particularly preferred embodiment, susceptibility of the individual
patient's
cancer and natural kill (NK) cells to the inhibitory effects of zosuquidar on
that patient's
P-gp expression and function is incorporated into the treatment regimen. Thus,
the cells
of interest are assessed as in one of the above-described methods both before
and after
exposure to zosuquidar. Such an assay can be considered a "zosuquidar
drugability"
assay to determine the potential for improved response to chemotherapy with
administration of zosuquidar. Similar methodology can be employed in providing
assays
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to determine drugability for other P-gp efflux inhibitors, and thus determine
the potential
for improved response to chemotherapy treatment regimens employing P-gp efflux
inhibitors.
Ranges of P-gp efflux pump activation and populations of cells have been
correlated to identify and stratify patients for future treatment with
enhanced clinical
outcomes. The diagnostic testing employed preferably includes assay methods as
described above, including immunophenotyping and cytogenetics of the cancer
cells, as
well as a diagnostic algorithm to relate the phenotypic and clinical data to
chemotherapeutic response potential.
While the techniques are especially preferred for use in administering
zosuquidar,
the techniques are applicable to any therapeutic drug that is a substrate for
P-gp efflux.
Such drugs include, but are not limited to, P-glycoprotein substrates;
anticancer drugs
(e.g., vinca alkaloids such as vinblastine and vincristine; anthracyclines
such as
doxorubicin, daunorubicin, epirubicin; anthracenes such as bisantrene and
mitoxantrone;
epipodophyllo-toxins such as etoposide and teniposide; and other anticancer
drugs such as
actinomyocin D, mithomycin C, mitramycin, methotrexate, docetaxel, etoposide
(VP- 16),
paclitaxel, docetaxel, and adriamycin); immunosuppressants (e.g., cyclosporine
A,
tacrolimus); steroids (e.g., dexamethasone, hydrocortisone, corticosterone,
triamcinolone,
aldosterone, methylprednisolone); antiepileptics (e.g., phenytoin);
antidepressants (e.g.,
citalopram, thioperidone, trazodone, trimipramine, ainitriptyline,
phenothiazines);
antipsychotics (fluphenazine, haloperidol, thioridazine, trimipramine); HIV
protease
inhibitors (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritqnavir,
saquinavir); calcium
blockers (e.g., bepridil, diltiazem, flunarizine, lomerizine, secoverine,
tamolarizine,
verapamil, nicardipine, prenylamine, fendiline); and cardiac drugs (e.g.,
digoxin,
diltiazem, verapamil, talinolol).
A P-gp diagnostic test exhibits clinical utility for a broad range of disease
states.
As discussed above, the drugs which are substrates of P-gp are quite varied as
are the
associated disease states. The use of a P-gp diagnostic test is best
exemplified in the
treatment of AML, where it has been demonstrated that the levels of P-gp
expression and
function are significantly related to response to the chemotherapy.
Preferred embodiments involve assessment on a patient-specific basis for
zosuquidar to modulate P-gp function in the cancer cell population. Thus, the
expression
and function of P-gp is assessed in the absence and presence of zosuquidar
with the
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patient's cancer and NK cells. A statistical algorithm to relate
immunophenotype of the
cancer cells, P-gp function and expression, cytogenetics, and certain clinical
variables
such as age to response potential to chemotherapy is used. This methodology
can be
applied to any form of cancer as related to chemotherapeutic agents which are
substrates
of P-gp as listed above.
The distribution of P-gp expression also has utility for a P-gp diagnostic
beyond
the oncology field. Affected organs can include the intestine, liver,
placenta, kidneys, and
blood brain barrier (BBB). The system of P-gp expression is believed to have
evolved to
eliminate or exclude toxins and metabolites. For unknown reasons,
hematopoietic and
lymphoid cells, such as myeloid, NK, and CD8 (cytotoxic T cells, CTL) cells
also express
P-gp. Disease states originating from these organs/cells can exhibit varied
responses to
therapy depending on the levels of P-gp expression. Moreover, P-gp is highly
expressed
by mucosal and luminal surfaces of organs which are involved in drug
absorption,
distribution, and excretion. Varied expression of P-gp can influence therapy
by
influencing drug pharmacokinetics in addition to pharmacodynamics. Therefore,
a P-gp
diagnostic test capable of stratifying patient populations according to
therapeutic response
potential has substantial clinical utility for a variety of disease
conditions.
For example, treatment of autoimmune, other inflammatory diseases, and organ
transplant with iminunosuppressive drugs, as well as with steroids, can be
optimized
using a P-gp diagnostic test. This is indicated by the enhanced expression of
MDR1 in
renal transplant patients who undergo graft rejection on cyclosporine. Other
exainples
include patients with rheumatoid arthritis, inflammatory bowel disease, and
systemic
lupus erythematosus. Varied expression of P-gp by the BBB can predict response
to
epilepsy drugs and other drugs used to treat mental disorders. Response
potential to
treatment of HIV and other viral therapeutics can also be optimized with a P-
gp
diagnostic test. Such a test can also find clinical utility in the prediction
of adverse
events, especially as related to the use of cardiovascular drugs. Such a test
is not currently
being conducted to predict prospectively response to therapy for this diverse
disease set.
The identification of tumor cells expressing P-gp or exhibiting positive P-gp
function involves a relationship between negative and positive control
situations.
Expression assays that use antibodies to detect P-gp expression include
controls of cells
either not treated with primary antibody, or cells treated with an antibody of
the same
isotype as that of the anti-P-gp reagent. At a cutoff where cells treated
under these latter
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conditions exhibited minimal positive reaction, e.g., 0-5% positive cells,
positive results
using the anti-P-gp antibody include 10% or more positive cells, typically 10-
25%
positive cells. Functional assays involve a ratio of P-gp dye accumulation or
efflux in the
presence and absence of a specific P-gp inhibitor such as zosuquidar. For the
accumulation assay format the amount of dye taken up by the cells in the
presence of
zosuquidar is divided by that amount of dye taken up by cells cultured without
zosuquidar. For the efflux assay format the amount of dye eliminated from the
cells in the
presence of zosuquidar is divided by that amount of dye eliminated without
zosuquidar in
the culture. Positive P-gp functional activity is inferred for ratios greater
than about 1:1.2,
typically ratios of from about 1:1.2 to about 1:1.5. For the efflux P-gp
functional assay,
ratios can be used, as well as % efflux and % inhibition of efflux by
zosuquidar or other
P-gp modulators. Positive P-gp functional activity is inferred by % efflux of
from about
30% to about 50%, and specific inhibition by a P-gp modulator is similarly
from about
30% to about 50%.
Chemotherapeutic Regimens Utilizing Zosuquidar and Mylotar~
In preferred embodiments, a P-gp expression or efflux pump activity diagnostic
is
conducted to provide information in treating AML patients or patients with
metastatic
breast cancer with zosuquidar in combination with Mylotarg. If the results of
the P-gp
expression or efflux pump activity diagnostic indicates positive P-gp
expression or efflux
pump activity, then treatment with zosuquidar (or another P-gp efflux
inhibitor) in
combination with Mylotarg is initiated. If the results of the P-gp expression
or efflux
pump activity diagnostic indicate negative P-gp expression or efflux pump
activity, then
zosuquidar is expected not to yield an improvement in clinical outcome and
another
treatment option not involving administration of a P-gp efflux inhibitor is
selected. In
relapsed AML patients, it is generally considered acceptable clinical practice
to wait for
P-gp expression or efflux pump activity test results before initiating a
treatment.
However, in certain embodiments it can be desirable to initiate treatment
before receiving
test results, and then reevaluate the desirability of continuing treatment,
depending upon
the test results. Most preferably, P-gp expression or efflux pump activity of
a sample both
in the presence and absence of the P-gp efflux inhibitor is compared, whereby
the P-gp
efflux that is inhibitable by the P-gp efflux inhibitor can be determined.
However, in
certain embodiments wherein P-gp expression or function status correlates with
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expectation of clinical success, it can be useful to determine P-gp expression
or efflux
pump activity at any point in time.
Mylotarg was approved in May 2000 for relapsed CD33-positive AML patients
over the age of 60. Mylotarg from Wyeth and Celltech is based on antibody-
targeted
chemotherapy. Mylotarg's highly specific antibody recognizes a cell-surface
molecule,
CD33, which is abundant on AML cells (>90%) but absent from norinal blood stem
cells,
the seeds from which normal blood and immune cells originate. The antibody is
linked to
calicheamicin, a potent chemotherapy agent. The antibody selectively targets
leukemic
blast cells and delivers calicheamicin to them. The chemical structure of
Mylotarg is
provided below.
n, average loading of
hP67.6 callicheamicln derivative
on antibody, is 2 to 3
moleslmole
NtJ
0 0 C-13 OH3
NHNA'A hif~=~ C7
G~#;~
~~~ O CH3 O NH .f, O
0
CHS 0 OCH3
0 OCM3 ON HN C3~
~O H
OCH3 CH2Cll x
Ft+~a O a {~
~}"~3 v("'SIr1 CH3
1 OCH,
0 n
There is a growing body of evidence to suggest that the calicheamicin
component
of Mylotarg is also an MDR substrate and subject to the P-gp efflux pump. In
several
studies, the cytotoxic effect of Mylotarg has been shown to be inversely
correlated with
the amount of P-gp present. Two MDR modulators, valspodar and the quinolone
derivative MS-209, have both been shown to reverse the resistance to Mylotarg
in P-gp
expressing CD33(+) leukemia cells and clinical studies are underway in
combination with
cyclosporine.
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The combination of zosuquidar, a highly specific and safe P-gp efflux
inhibitor, in
combination with Mylotarg or another calicheamicin-antibody conjugate is
effective for
treatment of relapsed AML. The effective dose of zosuquidar and the timing of
administration of zosuquidar and Mylotarg are critical to achieving improved
complete
remission rates and enhanced leukemia free and overall survival rates in the
relapsed
AML patient population. While the methods and formulations of preferred
embodiments
are especially preferred for treatment of relapsed AML patients, the methods
and
formulations can be adapted to other drugs and indications. For example, P-gp
efflux
inhibitors other than zosuquidar and/or chemotherapeutics other than Mylotarg
can be
administered according to the disclosed dosing regimens, or slightly modified
dosing
regimens. Likewise, the formulations and dosing regimens employing zosuquidar
and
Mylotarg can be employed in treating AML patients other than relapsed AML
patients, or
for other types of leukemia or other cancers that express P-gp, e.g., many
solid tumors,
lymphomas, bladder cancer, pancreatic cancer, ovarian cancer, liver cancer,
myeloma,
lymphocytic leukemia, breast cancer, and sarcoma.
Zosuquidar or certain other therapeutic agents can be administered in the form
of a
pharmaceutically acceptable salt, e.g., the trihydrochloride salt. The terms
"pharmaceutically acceptable salts" and "a pharmaceutically acceptable salt
thereof' as
used herein are broad terms and are used in their ordinary sense, including,
without
limitation, to refer to salts prepared from pharmaceutically acceptable, non-
toxic acids or
bases. Suitable pharmaceutically acceptable salts include metallic salts,
e.g., salts of
aluminum, zinc, alkali metal salts such as lithium, sodium, and potassium
salts, alkaline
earth metal salts such as calcium and magnesium salts; organic salts, e.g.,
salts of lysine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine (N-methylglucamine), procaine, and tris; salts of
free acids
and bases; inorganic salts, e.g., sulfate, hydrochloride, and hydrobromide;
and other salts
which are currently in widespread pharmaceutical use and are listed in sources
well
known to those of skill in the art, such as, for exainple, The Merck Index.
Any suitable
constituent can be selected to make a salt of zosuquidar or other therapeutic
agents
discussed herein, provided that it is non-toxic and does not substantially
interfere with the
desired activity. In addition to salts, pharmaceutically acceptable precursors
and
derivatives of the compounds can be employed. Pharmaceutically acceptable
amides,
lower alkyl esters, and protected derivatives can also be suitable for use in
compositions
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and methods of preferred embodiments. Also suitable for administration are
selected
therapeutic agents in hydrated form, selected enantiomeric forms of certain
therapeutic
agents, racemic mixtures of certain therapeutic agents, and the like.
Contemplated routes of administration include topical, oral, subcutaneous,
parenteral, intradermal, intramuscular, intraperitoneal, and intravenous.
However, it is
particularly preferred to administer zosuquidar and/or Mylotarg in intravenous
form. The
combination or individual components can be in any suitable solid or liquid
form. A
particularly preferred form comprises a lyophilized form that is reconstituted
for
intravenous administration.
Zosuquidar and/or other therapeutic agents can be formulated into liquid
preparations for, e.g., oral, nasal, anal, rectal, buccal, vaginal, peroral,
intragastric,
mucosal, perlingual, alveolar, gingival, olfactory, or respiratory mucosa
administration.
Suitable forms for such administration include suspensions, syrups, and
elixirs. If nasal
or respiratory (mucosal) administration is desired (e.g., aerosol inhalation
or insufflation),
compositions may be in a form and dispensed by a squeeze spray dispenser, pump
dispenser or aerosol dispenser. Aerosols are usually under pressure by means
of a
hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose
having a
particular particle size.
The pharmaceutical compositions containing zosuquidar and/or other therapeutic
agents are preferably isotonic with the blood or other body fluid of the
patient. The
isotonicity of the compositions can be attained using sodium tartrate,
propylene glycol or
other inorganic or organic solutes. Sodium chloride is particularly preferred.
Buffering
agents can be employed, such as acetic acid and salts thereof, citric acid and
salts thereof,
boric acid and salts thereof, and phosphoric acid and salts thereof.
Parenteral vehicles
include sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride,
lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and
nutrient
replenishers, electrolyte replenishers (such as those based. on Ringer's
dextrose), and the
like.
Viscosity of the pharmaceutical compositions can be maintained at a selected
level
using a pharmaceutically acceptable thickening agent. Methylcellulose is
preferred
because it is readily and economically available and is easy to work with.
Other suitable
thickening agents include, for example, xanthan gum, carboxymethyl cellulose,
hydroxypropyl cellulose, carbomer, and the like. The preferred concentration
of the
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thickener can depend upon the thickening agent selected. An amount is
preferably used
that can achieve the selected viscosity. Viscous compositions are normally
prepared from
solutions by the addition of such thickening agents.
A pharmaceutically acceptable preservative can be employed to increase the
shelf
life of the pharmaceutical compositions. Benzyl alcohol can be suitable,
although a
variety of preservatives including, for example, parabens, thimerosal,
chlorobutanol, and
benzalkonium chloride can also be employed. A suitable concentration of the
preservative is typically from about 0.02% to about 2% based on the total
weight of the
composition, although larger or smaller amounts can be desirable depending
upon the
agent selected.
The zosuquidar and/or other therapeutic agents can be in admixture with a
suitable
carrier, diluent, or excipient such as sterile water, physiological saline,
glucose, and the
like, and can contain auxiliary substances such as wetting or emulsifying
agents, pH
buffering agents, gelling or viscosity enhancing additives, preservatives,
flavoring agents,
colors, and the like, depending upon the route of administration and the
preparation
desired. See, e.g., standards texts such as "Remington: The Science and
Practice of
Pharmacy", Lippincott Williams & Wilkins; 20th edition (June 1, 2003) and
"Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18th and 19th editions
(December 1985, and June 1990, respectively). Such preparations can include
'complexing agents, metal ions, polymeric compounds such as polyacetic acid,
polyglycolic acid, hydrogels, dextran, and the like, liposomes,
microemulsions, micelles,
unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
Suitable lipids
for liposomal formulation include, without limitation, monoglycerides,
diglycerides,
sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
The presence of
such additional components can influence the physical state, solubility,
stability, rate of in
vivo release, and rate of in vivo clearance, and are thus chosen according to
the intended
application, such that the characteristics of the carrier are tailored to the
selected route of
administration.
For oral administration, the zosuquidar and/or other therapeutic agents can be
provided as a tablet, aqueous or oil suspension, dispersible powder or
granule, emulsion,
hard or soft capsule, syrup, or elixir. Compositions intended for oral
administration can
be prepared according to any method known in the art for the manufacture of
pharmaceutical compositions and can include one or more of the following
agents:
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sweeteners, flavoring agents, coloring agents and preservatives. Aqueous
suspensions can
contain the active ingredient in admixture with excipients suitable for the
manufacture of
aqueous suspensions.
Formulations for oral administration can also be provided as hard gelatin
capsules,
wherein the zosuquidar and/or other active ingredients are mixed with an inert
solid
diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft
gelatin
capsules. In soft capsules, the active ingredients can be dissolved or
suspended in suitable
liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty
oils, liquid
paraffin, or liquid polyethylene glycols. Stabilizers and microspheres
formulated for oral
administration can also be used. Capsules can include push-fit capsules made
of gelatin,
as well as soft, sealed capsules made of gelatin and a plasticizer, such as
glycerol or
sorbitol. The push-fit capsules can contain the active ingredients in
admixture with fillers
such as lactose, binders such as starches, and/or lubricants such as talc and
magnesium
stearate and, optionally, stabilizers.
Tablets can be uncoated or coated by known methods to delay disintegration and
absorption in the gastrointestinal tract and thereby provide a sustained
action over a
longer period of time. For example, a time delay material such as glyceryl
monostearate
can be used. When administered in solid form, such as tablet form, the solid
form
typically comprises from about 0.001 wt. % or less to about 50 wt. % or more
of active
ingredient(s) including zosuquidar and/or other therapeutic agents, preferably
from about
0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7,
0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, or 45 wt. %.
Tablets can contain the zosuquidar and/or other therapeutic agents in
admixture
with non-toxic pharmaceutically acceptable excipients including inert
materials. For
example, a tablet can be prepared by compression or molding, optionally, with
one or
more additional ingredients. Compressed tablets can be prepared by compressing
in a
suitable machine the active ingredients in a free-flowing form such as powder
or granules,
optionally mixed with a binder, lubricant, inert diluent, surface active or
dispersing agent.
Molded tablets can be made by molding, in a suitable machine, a mixture of the
powdered
compound moistened with an inert liquid diluent.
Preferably, each tablet or capsule contains from about 10 mg or less to about
1,000
mg or more of each of zosuquidar and/or other therapeutic agents, more
preferably from
about 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300,
350, 400, 450,
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500, 550, 600, 650, 700, 750, 800, or 900 mg. Most preferably, tablets or
capsules are
provided in a range of dosages to permit divided dosages to be administered. A
dosage
appropriate to the patient and the number of doses to be administered daily
can thus be
conveniently selected. While in certain embodiments it can be preferred to
incorporate
the zosuquidar and any other therapeutic agent employed in combination
therewith in a
single tablet or other dosage form, in certain embodiments it can be desirable
to provide
the zosuquidar and other therapeutic agents in separate dosage forms, e.g.,
zosuquidar in a
dosage form separate from other agents(s). Combinations of dosage forms can
also be
employed, e.g., oral and intravenous.
Suitable inert materials include diluents, such as carbohydrates, mannitol,
lactose,
anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the
like, and
inorganic salts such as calcium triphosphate, calcium phosphate, sodium
phosphate,
calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride.
Disintegrants or granulating agents can be included in the formulation, for
example,
starches such as corn starch, alginic acid, sodium starch glycolate,
Amberlite, sodium
carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange
peel, acid
carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic
exchange
resins, powdered gums such as agar, karaya, and tragacanth, and alginic acid
and salts
thereof.
Binders can be used to form a hard tablet. Binders include materials from
natural
products such as acacia, tragacanth, starch, gelatin, methyl cellulose, ethyl
cellulose,
carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose,
and the
like.
Lubricants, such as stearic acid and magnesium or calcium salts thereof,
polytetrafluoroethylene, liquid paraffin, vegetable oils, waxes, sodium lauryl
sulfate,
magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica,
hydrated
silicoaluminate, and the like can be included in tablet formulations.
Surfactants can also be employed, for example, anionic detergents such as
sodium
lauryl sulfate, dioctyl sodium sulfosuccinate, and. dioctyl sodium sulfonate,
cationic
detergents such as benzalkonium chloride and benzethonium chloride, and/or
nonionic
detergents such as polyoxyethylene hydrogenated castor oil, glycerol
monostearate,
polysorbates, sucrose fatty acid ester, methyl cellulose, and carboxymethyl
cellulose.
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Controlled-release formulations can be employed wherein the zosuquidar and/or
other therapeutic agents are incorporated into an inert matrix that permits
release by either
diffusion or leaching mechanisms. Slowly degenerating matrices can also be
incorporated
into the formulation. Other delivery systems can include timed release,
delayed release,
or sustained release delivery systems. Nanoparticulate systems or
nanoparticulate forms
of the active ingredients can advantageously be employed in certain
embodiments.
Coatings can be used, for example, nonenteric materials such as methyl
cellulose,
ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose,
hydroxypropyl
cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone,
polyethylene glycols, and enteric materials such as phthalic acid esters.
Dyestuffs and
pigments can be added for identification or to characterize different
combinations of
active compound doses
When administered orally in liquid form, a liquid carrier such as water,
petroleum,
oils of animal or plant origin such as peanut oil, mineral oil, soybean oil,
or sesame oil, or
synthetic oils can be added to the zosuquidar and/or other therapeutic agents.
Physiological saline solution, dextrose, other saccharide solutions, and
glycols such as
etliylene glycol, propylene glycol, and polyethylene glycol are also suitable
liquid carriers.
The pharmaceutical compositions can also be in the form of oil-in-water
emulsions. The
oily phase can be a vegetable oil, such as olive or arachis oil, a mineral oil
such as liquid
paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-
occurring
gums such as gum acacia and gum tragacanth, naturally occurring phosphatides,
such as
soybean lecithin, esters or partial esters derived from fatty acids and
hexitol anhydrides,
such as sorbitan mono-oleate, and condensation products of these partial
esters with
ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions
can also
contain sweetening and flavoring agents.
Pulmonary delivery of zosuquidar and/or other therapeutic agents can also be
employed. The zosuquidar and/or other therapeutic agents are delivered to the
lungs
while inhaling and traverse across the lung epithelial lining to the blood
stream. A wide
range of mechanical devices designed for pulmonary delivery of therapeutic
products can
be einployed, including but not limited to nebulizers, metered dose inhalers,
and powder
inhalers, all of which are familiar to those skilled in the art. These devices
employ
formulations suitable for the dispensing of zosuquidar and/or other
therapeutic agents.
Typically, each formulation is specific to the type of device employed and can
involve the
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use of an appropriate propellant material, in addition to diluents, adjuvants,
and/or carriers
useful in therapy.
The zosuquidar and/or other therapeutic agents are advantageously prepared for
pulmonary delivery in particulate form with an average particle size of from
0.1 m or
less to 10 m or more, more preferably from about 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, or 0.9
m to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,
7.5, 8.0, 8.5, 9.0, or
9.5 m. Pharmaceutically acceptable carriers for pulmonary delivery of
zosuquidar
and/or other therapeutic agents include carbohydrates such as trehalose,
mannitol, xylitol,
sucrose, lactose, and sorbitol. Other ingredients for use in formulations can
include
dipalmitoylphosphatidylcholine (DPPC), 1,2-sn-dioleoylphosphatidylcholine
(DOPE),
disteroylphosphatidylcholine (DSPC), and dioleoylphosphatidyl-choline (DOPC).
Natural or synthetic surfactants can be used, including polyethylene glycol
and dextrans,
such as cyclodextran. Bile salts and other related enhancers, as well as
cellulose and
cellulose derivatives, and amino acids can also be used. Liposomes,
microcapsules,
microspheres, inclusion complexes, and other types of carriers can also be
employed.
Pharmaceutical formulations suitable for use with a nebulizer, either jet or
ultrasonic, typically comprise the zosuquidar and/or other therapeutic agents
dissolved or
suspended in water at a concentration of about 0.01 mg or less to 100 mg or
more of
zosuquidar and/or other therapeutic agents per mL of solution, preferably from
about 0.1,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg per mL of solution to about 15, 20, 25,
30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulation can
also include
a buffer and a simple sugar (e.g., for protein stabilization and regulation of
osmotic
pressure). The nebulizer formulation can also contain a surfactant, to reduce
or prevent
surface induced aggregation of the zosuquidar and/or other therapeutic agents
caused by
atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose inhaler device generally comprise a
finely divided powder containing the active ingredients suspended in a
propellant with the
aid of a surfactant. The propellant can include conventional propellants, such
as
chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, and
hydrocarbons.
Preferred propellants include trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, and combinations
thereof. Suitable
surfactants include sorbitan trioleate, soya lecithin, and oleic acid.
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Formulations suitable for dispensing from a powder inhaler device typically
comprise a finely divided dry powder containing zosuquidar and/or other
therapeutic
agents, optionally including a bulking agent, such as lactose, sorbitol,
sucrose, mannitol,
trehalose, or xylitol in an amount that facilitates dispersal of the powder
from the device,
typically from about 1 wt. % or less to 99 wt. % or more of the formulation,
preferably
from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65,
70, 75, 80,
85, or 90 wt. % of the formulation.
When zosuquidar and/or other therapeutic agents are administered by
intravenous,
cutaneous, subcutaneous, parenteral, or other injection, they are preferably
in the form of
pyrogen-free, parenterally acceptable aqueous solution or oleaginous
suspension.
Suspensions can be formulated according to methods well known in the art using
suitable
dispersing or wetting agents and suspending agents. The preparation of
acceptable
aqueous solutions with suitable pH, isotonicity, stability, and the like, is
within the skill in
the art. A preferred pharmaceutical composition for injection preferably
contains an
isotonic vehicle such as 1,3-butanediol, water, isotonic sodium chloride
solution, Ringer's
solution, dextrose solution, dextrose and sodium chloride solution, lactated
Ringer's
solution, or other vehicles as are known in the art. In addition, sterile
fixed oils can be
employed conventionally as a solvent or suspending medium. For this purpose,
any bland
fixed oil can be employed, including synthetic monoglycerides and
diglycerides. In
addition, fatty acids stich as oleic acid can likewise be used in the
formation of injectable
preparations. The pharmaceutical compositions can also contain stabilizers,
preservatives, buffers, antioxidants, and other additives known to those of
skill in the art.
The duration of the injection can be adjusted depending upon various factors,
and
can comprise a single injection administered over the course of a few seconds
or less to 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 26, 28, 30,
32, 34, 36, 40, 44, 48, 54, 60, 66, 72, 78; 84, 90, or 96 hours or more of
continuous
intravenous administration.
The zosuquidar and/or other therapeutic agents can be administered
systemically
or locally, via a liquid or gel, or as an implant or device.
The coinpositions of the prefeiTed embodiments can additionally employ adjunct
components conventionally found in pharmaceutical compositions in their art-
established
fashion and at their art-established levels. Thus, for example, the
compositions can
contain additional compatible pharmaceutically active materials for
combination therapy
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(such as supplemental P-gp inhibitors, chemotherapeutic agents, and the like),
or can
contain materials useful in physically formulating various dosage forms of the
preferred
embodiments, such as excipients, dyes, perfumes, thickening agents,
stabilizers,
preservatives and antioxidants.
The zosuquidar and/or other therapeutic agents can be provided to an
administering physician or other health care professional in the form of a
kit. The kit is a
package which houses one or more containers which contain zosuquidar and/or
additional
therapeutic agents in suitable form and instructions for administering the
pharinaceutical
composition to a subject. The kit can optionally also contain one or more
additional
I Q therapeutic agents. The kit can optionally contain one or more assays or
diagnostic tools
and instructions for use, e.g., a diagnostic to measure efflux pump activity
or P-gp
expression or function. For example, a kit containing a single composition
comprising
zosuquidar with one or more chemotherapeutic agents can be provided, or
separate
pharmaceutical coinpositions containing zosuquidar and other therapeutic
agents can be
provided. The kit can also contain separate doses of zosuquidar and/or other
therapeutic
agents for serial or sequential administration. The kit can contain suitable
delivery
devices, e.g., syringes, inhalation devices, and the like, along with
instructions for
administrating zosuquidar and/or other therapeutic agent. The kit can
optionally contain
instructions for storage, reconstitution (if applicable, e.g., for a
lyophilized form
reconstituted for intravenous administration), and administration of any or
all therapeutic
agents included. The kits can include a plurality of containers reflecting the
number of
administrations to be given to a subject. In a particularly preferred
embodiment, a kit for
the treatment of AML is provided that includes both zosuquidar, a
chemotherapeutic
agent, and one or more diagnostics or instructions for conducting one or more
diagnostics
for determining P-gp expression and/or efflux pump activity. In a particularly
preferred
embodiment, the kit includes a "zosuquidar drugable" assay, as previously
described.
Zosuquidar and a therapeutic agent that is a substrate for P-gp efflux can be
administered to patients suffering from AML prior to confirmation of P-gp
expression or
function, or to AML patients other than relapse AML patients. However, such
therapy is
preferably administered to relapsed AML patients. The administration route,
amount
administered, and frequency of administration can vary depending on the age of
the
patient, status as relapsed or newly diagnosed AML patient, and severity of
the condition
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Contemplated amounts of Mylotarg for intravenous administration to treat
relapsed AML are from about 10 mg/day or less to about 1000 mg/day or more
administered on one, two, or more separate days. The dosage is preferably
administered
intravenously at a rate of about 1 mg/ma or less to about 10 mg/mz or more
continuously
over the course of about 2, 3, or 4 hours to about 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, or 24 hours, more preferably over the course of
about 2 hours to
about 6 hours; however, administration at a rate of 5 mg/m2, 7 mg/m2, or 9
mg/m2 over
about 2 hours is particularly preferred. Preferably, doses of Mylotarg are
administered on
Day 1 and Day 15 of the treatment regimen. However, in certain embodiments,
the
second dose can be adininistered on Day 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,
17, 18, 19,
20, 21, or 22, or another day of the treatment regimen. Other dosing regimens
include
administering three doses total over a week.
Contemplated amounts of zosuquidar for intravenous administration to treat
relapsed AML are from about 400 mg/day or less to about 1,600 mg/day or more,
preferably from about 500, 600, or 700 mg/day to about 900, 1000, 1100, 1200,
1300,
1400, or 1500 mg/day, and most preferably from about 500 mg/day to about 800
mg/day.
It is generally preferred to start the infusion of zosuquidar from about 2
hours or less to
about 6 hours or more prior to the administration of Mylotarg. In the course
of a
treatment regimen, the zosuquidar is preferably administered on two, three, or
four
separate days. The dosage is preferably administered in intravenously
continuously over
the course of about 6 to 90 hours, more preferably over the course of 12, 18,
24, 30, 36, or
42 hours to about 54, 60, 66, 72, 78, or 84 hours, most preferably over about
24 hours, 48
hours, or 72 hours, depending upon the treatment regimen. Preferably the
zosuquidar is
administered on Day 1 of the treatment regimen. In certain embodiments,
additional
zosuquidar is administered on Day 2, on Days 2 and 3, or on Days 2, 15, and
16.
However, in certain embodiments, one, two, or three or more additional doses
can be
administered on other days of the treatment regimen.
Table 1 provides various dosing regimes that can be used in treating relapsed
AML.
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Table 1.
Dose Mylotarg Zosuquidar
Level
-1 * 5 mg/m2 IV over 4 hr Day 1 and 15 800 mg/day continuous IV over 24 hr
Day 1 and 15
1 5 mg/m2 IV over 4 hr Day 1 and 15 800 mg/day continuous IV over 48 hr
Day 1&2 and 15&16
2 7 mg/m2 IV over 4 hr Day 1 and 15 800 mg/day continuous IV over 48 hr
Day 1&2 and 15&16
3 9 mg/m2 IV over 4 hr Day 1 and 15 800 mg/day continuous IV over 48 hr
Day 1&2 and 15&16
4 9 mg/m2 IV over 4 hr Day 1 and 15 800 mg/day continuous IV over 72 hr
Day 1-3 and 15-17
* Only if level 1 has a dose limiting toxicity (DLT).
Table 2 provides alternative dosing regimes that can be used in treating
relapsed
AML.
Table 2.
Dose Mylotarg Zosuquidar
Level
-1 * 5 mg/m2 IV over 6-24 hr Day 1 and 500-700 mg/day continuous IV over 24
hr Day l and 15
1 5 mg/m2 IV over 6-24 hr Day 1 and 500-700 mg/day continuous IV over 48
15 hr Day 1&2 and 15&16
2 7 mg/m2 IV over 6-24 hr Day 1 and 500-700 mg/day continuous IV over 48
15 hr Day 1&2 and 15&16
3 9 mg/m2 IV over 6-24 hr Day 1 and 500-700 mg/day continuous IV over 48
15 hr Day l&2 and 15&16
4 9 mg/ma IV over 6-24 hr Day 1 and 500-700 mg/day continuous IV over 72
15 hr Day l-3 and 15-17
* Only if level 1 has a dose limiting toxicity (DLT).
10 A clinical study was conducted to determine the efficacy of Mylotarg in the
treatment of relapsed AML. It was determined that the rate of complete
remission (CR +
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CRp) for P-gp negative patients treated with Mylotarg was 64% (N=36). In
contrast, the
rate of complete remission for P-gp positive patients was only 9% (N=22). This
indicates
that P-gp efflux plays an important role in survival rates for relapsed AML,
and further
indicates that inhibition of P-gp efflux, e.g., by also administering
zosuquidar or another
P-gp efflux inhibitor, has the potential to significantly improve response
rates in P-gp
positive patients. The diagnostic and assay methods described herein are
therefore useful
in treating relapsed AML. Likewise, a diagnostic or assay to determine P-gp
expression
or function or efflux pump activity can be useful in devising treatment
regimens for other
cancers, such as metastatic breast cancer, that also exhibit P-gp expression.
Chemotherapeutic Regimens Utilizing Zosuquidar Daunorubicin, and Cytarabine
In preferred embodiments, a P-gp expression or efflux pump activity diagnostic
is
conducted to provide information in treating newly diagnosed AML patients with
zosuquidar in combination with daunorubicin and cytarabine. In newly diagnosed
AML
patients, it is generally not considered acceptable clinical practice to wait
for P-gp
expression or efflux pump activity test results before initiating a treatment.
Accordingly,
treatment is initiated immediately after diagnosis. When test results become
available, the
desirability of continuing treatment can be evaluated, depending upon the test
results.
Typically, when the results of the P-gp expression or efflux pump activity
diagnostic
indicate negative P-gp expression, then treatment with a P-gp efflux inhibitor
is
discontinued because administration of the drug is not expected to contribute
to an
improved clinical outcome. Preferably, P-gp expression or function or efflux
pump
activity is deterinined both in the presence and the absence the P-gp efflux
inhibitor to
determine the P-gp expression that is inhibitable by the P-gp efflux
inhibitor.
Daunorubicin is an antibiotic chemotherapy treatment that is widely used to
treat
acute myeloid leulcemia and acute lymphocytic leukemia. It is produced by the
bacteria
Streptomyces coeruleorubidis and was approved by the FDA as a first line
therapy
treatment for leukemia in 1998. Daunorubicin is typically administered
intravenously. It
is marketed under the brand names Cerubidine, DaunoXome, and Liposomal
daunorubicin. Daunorubicin has the following structure:
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00 H OCH3
C7H
OM e 0 OH 0- H---CHz-CHM..Cil---CH-CH.v'HCS
Cytarabine is a deoxycytidine analogue, cytosine arabinoside (ara-C), which is
metabolically activated to the triphosphate nucleotide (ara-CTP), which acts
as a
competitive inhibitor of DNA polymerase and produces S phase-specific
cytotoxicity. It
is used as an antineoplastic, generally as part of a combination chemotherapy
regimen, in
the treatment of acute' lymphocytic and acute myelogenous leulcemia, the blast
phase of
chronic myelogenous leukemia, erythroleukemia, and non-Hodgkin's lymphoma. It
is
typically administered intravenously and subcutaneously, and for the
prophylaxis and
treatment of meningeal leukemia, administered intrathecally. Cytarabine has
the
following structure:
H
0N
HCH',
OH
The combination of zosuquidar, the antibiotic chemotherapeutic daunorubicin,
and
the antineoplastic cytarabine, is effective for treatment of newly diagnosed
AML. The
effective dose of zosuquidar and the timing of administration of zosuquidar,
daunorubicin, and cytarabine are critical to achieving improved coinplete
remission rates
and enhanced leukemia free survival rates in the newly diagnosed AML patient
population. While the methods and formulations of preferred embodiments are
especially
preferred for treatment of newly diagnosed AML patients, the methods and
formulations
can be adapted to other drugs and indications. For example, P-gp efflux
inhibitors other
than zosuquidar and/or chemotherapeutics other than daunorubicin and
cytarabine can be
administered according to the disclosed dosing regimens, or slightly modified
dosing
regimens. Likewise, the formulations and dosing regimens employing zosuquidar,
daunorubicin, and cytarabine can be employed in treating AML patients other
than newly
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diagnosed AML patients, or for treating other types of leukemia or other
cancers that
exhibit P-gp expression, as discussed above.
Zosuquidar, daunorubicin, and cytarabine can be formulated as described above
for zosuquidar and Mylotarg, and can be included in kits, also as described
above.
The zosuquidar, daunorubicin, and/or cytarabine can be to patients suffering
from
AML prior to confirmation of the P-gp expression or function, or to AML
patients other
than newly diagnosed AML patients (e.g., relapsed AML patients). However,
therapy is
preferably administered to newly diagnosed AML patients. The administration
route,
amount administered, and frequency of administration can vary depending on the
age of
the patient, status as relapsed or newly diagnosed AML patient, and severity
of the
condition.
Contemplated amounts of zosuquidar for intravenous administration to treat
newly
diagnosed AML are from about 400 mg/day or less to about 1,600 mg/day or more,
preferably from about 500, 600, or 700 mg/day to about 900, 1000, 1100, 1200,
1300,
1400, or 1500 mg/day, and most preferably 700 mg/day. In the course of a
treatment
regimen, the zosuquidar is preferably administered on two, three, or four
separate days.
The dosage is preferably administered in intravenously continuously over the
course of
about 6 to about 90 hours, more preferably over the course of about 12, 18,
24, 30, 36, or
42 hours to about 54, 60, 66, 72, 78, or 84 hours, most preferably over about
24 hours, 48
hours, or 72 hours, depending upon the treatment regimen. Preferably the
zosuquidar is
administered on Day 1 of the treatment regimen. In certain embodiments,
additional
zosuquidar is administered on Day 2, on Days 2 and 3, or on Days 2, 15, and
16.
However, in certain embodiments, one, two, or three or more additional doses
can be
administered on other days of the treatment regimen.
Contemplated amounts of daunorubicin for intravenous administration to treat
newly diagnosed AML are from about 10 mg/m2/day or less to about 100
ing/m2/day or
more administered at initiation of zosuquidar infusion or up to about 1, 2, 3,
4, 5, or 6 or
more hours after initiation of zosuquidar infusion. The dosage is preferably
administered
intravenously at a rate of about 25 mg/m2/day or less to about 90 mg/m2/day or
more,
preferably about 30, 35, or 40 mg/m2/day or less to about 50, 55, 60, 65, 70,
75, 80, or 85
mg/m2 /day, and most preferably about 45 mg/mz/day continuously over the
course of
about 2 or 2.5 days to about 3.5 or 4 days, preferably about 3 days.
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Contemplated amounts of cytarabine for intravenous administration to treat
newly
diagnosed AML patients are from about 10 mg/day or less to about 3,000 mg/day
or more
administered at initiation of zosuquidar infusion or after initiation of
zosuquidar infusion.
The dosage is preferably administered intravenously at a rate of about 50
mg/m2/day or
less to about 200 mg/m2/day or more, preferably 60, 70, 80, or 90 mg/m2/day or
less to
about 110, 120, 130, 140, 150, 160, 170, 180, or 190 ing/m2/day, and most
preferably
about 100 ing/m2/day continuously over the course of about 1, 2, 3, 4, 5, or 6
days up to
about 8, 9, or 10 days or more, preferably over about 7 days.
A particularly preferred dosing regimen for newly diagnosed AML includes
continuous intravenous administration of 550 mg of zosuquidar over 6 hours (3
days),
continuous intravenous administration of cytarabine at a rate of 100 mg/m2/day
(7 days),
and intravenous administration of daunorubicin at a dose of 45 mg/m2/day (3
days),
wherein infusion of daunorubicin is started 1 hour after initiation of
zosuquidar infusion.
Another particularly preferred dosing regimen includes continuous intravenous
administration (preferably about 1 to 24 hours in duration, more preferably
about 6 to 24
hours in duration, most preferably about 24 hours in duration) of 500 to 700
mg/day of
zosuquidar (3 days), continuous intravenous administration of cytarabine at a
rate of 100
mg/m2/day (7 days), and intravenous administration of daunorubicin at a dose
of 45
mg/m2/day (3 days), wherein infusion of daunorubicin is started 1 to 4 hours
after
initiation of zosuquidar infusion. While in the above described embodiments
infusion of
daunorubicin is started after a specified time period has lapsed after
initiation of
zosuquidar infusion, in other embodiments other start times can be preferred,
e.g.,
immediately after or during initiation of zosuquidar infusion up to about 1,
2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, or more hours after initiation of zosuquidar infusion.
While the above methods of the preferred embodiments have been discussed
primarily in connection with the treatment of AML, the methods are also
particularly
efficacious when P-gp substrates are administered as chemotherapeutic agents
in the
treatment of other malignancies exhibiting some degree of P-gp expression. For
example,
such malignancies can include lymphomas, bladder cancer, pancreatic cancer,
ovarian
cancer, liver cancer, myeloma, lymphocytic leukemia, sarcoma, metastatic
breast cancer,
and most solid tumors. Chemotherapeutic agents that are P-gp substrates
include, but are
not limited to, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin,
idarubicin,
mitoxantrone), vincas (e.g., vincristine, vinblastine, vinorelbine,
vindesine),
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Topoisomerase-Il inhibitors (e.g., etoposide, teniposide), taxanes (e.g.,
paclitaxel,
docetaxel), and others (e.g., Gleevec, Mylotarg, dactinomycin, mithramycin).
Efflux Assay
A commonly used method for the detection of MDR1 functional activity is
referred to as the "efflux assay." In this assay the tumor cells are incubated
with a
fluorescent dye such as DiOC2 or Rhodamine 123 to allow for accumulation of
the dye.
The cells are then washed, a fraction of the cells are measured for dye
content
immediately or are placed on ice to obtain a baseline dye uptake control, and
the
remaining cells are recultured in the presence and absence of an MDR1
inhibitor, such as
zosuquidar. Since zosuquidar inhibits MDR1, the dye will be retained by such
treated
cells, and with cells cultured in the absence of zosuquidar the dye will be
eliminated
(effluxed). Retention and elimination of the dye is quantified by flow
cytometry. There is
a second way to detect retention or elimination of the dye and it is called
the
"accumulation assay." The cells in this assay are incubated with dye in the
presence and
absence of an MDR1 inhibitor such as zosuquidar. At the end of the incubation
period,
the cells are washed and assessed for retention of dye by flow cytometry. The
standard
method for detection of MDR1 activity is by the efflux assay. As shown herein,
the
accumulation assay is superior to the standard efflux assay for the assessment
of MDR1
status in cancer patients. However, the efflux assay can yield substantially
improved
signal to noise if the cells are allowed to accumulate dye in the presence of
an MDR1
inhibitor such as zosuquidar.
Identification of the Distinct Advantage of the Accumulation Assay Over the
Standard Efflux Assa s~g K562/R7 MDR1-positive and K562 MDR1-negative Cell
Lines
K562/R7 cells express MDR1 as a consequence of selection in the presence of
doxorubicin. The parental K562 cell line does not express appreciable MDR1.
DiOC2
was the dye selected as the surrogate fluorescent marker for MDR1 substrate
chemotherapeutic agents. The accumulation assay involved cells cultured at 5 x
105
cells/ml, 0.5 ml/ culture volume, 60 ng/ml DiOC2, and incubation at 37 C for
at least 30
and not longer than 90 minutes. The efflux assay involved culturing cells with
60 ng/ml
DiOC2, washing the cells and reculturing as described for the accumulation
assay. The
cells are then analyzed for fluorescence by flow cytometry.
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Figure 1 presents typical histograms of K562/R7. The upper panel shows
autofluorescence, i.e., cells cultured in the absence of DiOC2. K562/R7 cells
exhibit such
strong MDR1 activity and, as shown in the middle panel, accumulated minimal
dye in the
absence of zosuquidar. However, the cells could be forced to accumulate dye if
cultured
in the presence of zosuquidar (bottom panel). This illustrates the differences
between the
typical efflux and accumulation assays. In the typical efflux assay, the
amount of dye
ultimately loaded into the cells reflects the equilibrium between accumulation
and efflux
processes. In the accumulation assay with zosuquidar present, the amount of
dye
ultimately loaded into the cells reflects only influx since efflux is
inhibited.
The data are typically expressed as ratios of mean fluorescence intensity
(MFI) of
zosuquidar-treated cultures divided by the MFI of untreated cultures. In the
accumulation
assay, the inhibitory effects of zosuquidar on K562/R7 MDR1 function were
clearly
evident as indicated by' the inhibition ratio of 15.6. In the efflux assay,
assuming
complete elimination of the dye to autofluorescence levels, a ratio of 1.55
would be
observed, which is considered dim or negative MDRI activity with the standard
efflux
assay. Thus, as will be addressed subsequently, it is quite possible that
leukemia cells
with very high MDR1 activity could similarly be misclassified with the
standard efflux
assay.
The Distinct Advantage of the Accumulation Assay over the Standard Efflux
Assay Can Be Observed with Leukemia cells
A coinparison was conducted between the accumulation and efflux assays with
leukemia cells. Figure 2 presents a graphic depiction of the data. Negative or
"dim"
results are indicated by samples with ratios to the left of the vertical line
at a ratio of 1.55.
A number of samples exhibited comparable ratios in the two assays. However,
some
samples exhibited what would be considered dim or negative MDR1 functional
activity in
the efflux assay, but rather substantial activity in the accumulation assay.
While not
wishing to be bound by theory, a possible explanation for this observation
could be that in
the accumulation assay some highly active MDR1-positive cells, like K562/R7
cells
(referring to Figure 1), can be forced to accumulate dye provided that
zosuquidar is
present. In the efflux assay, where zosuquidar is not present during dye
loading, these
highly active cells would not accumulate enough dye to be identified as
effluxing during
the secondary efflux stage of that assay.
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Figure 3 presents an example of these phenomena in a leukemia cell sample. The
cells were loaded for the efflux assay and eitlier placed on ice (upper left
panel, baseline
control) or allowed to efflux in the absence (middle left panel) or presence
of zosuquidar
(lower left panel). The efflux ratio was 2.6. The maximum amount of dye in the
loaded
cells yielded a MFI = 121. However, as shown in the lower right panel, loading
in the
presence of zosuquidar (accumulation) yielded a MFI = 907 and a ratio = 7.5.
The data
can be interpreted as demonstrating, as with K562/R7 cells, that leukemia
cells with high
MDR1 activity only accumulate substantial amounts of dye when cultured in the
presence
of an MDRl inhibitor.
The Use of Zosuquidar during Dye Loading Can Yield a Highly Sensitive Efflux
Assay
Figure 4 presents the efflux characteristics of K562/R7 cells which had been
loaded with 40 ng/ml DiOC2 in the presence of 200 ng/ml zosuquidar for 60
minutes.
The loaded and washed cells were cultured at 37 C in 0.5 ml lacking or
containing the
indicated concentrations of drug for the indicated amount of time. Control
cells were
placed on ice immediately after loading and, as indicated by the symbol to the
far right at
MFI = 1172, the cells had been loaded with dye. Incubation (efflux) in the
presence of
zosuquidar inhibited dye release in a concentration-dependent manner which was
basically independent of incubation period. Significantly, in contrast to the
low efflux
ratio (1.55) illustrated in Figure 1 using the standard efflux methods,
loading cells in the
presence of zosuquidar yielded an efflux assay with a high efflux ratio =
26.8.
Discussion of Results
As shown herein, the standard efflux method suffers from a lack of sensitivity
when leukemia cells with very high MDR1 activity are tested. The amount of dye
present
in cells to be tested in the standard efflux assay reflects the equilibrium
between
accumulation and efflux during the loading period. The poor sensitivity of the
standard
efflux assay results from the high efflux rate of some cancer samples yielding
poorly
loaded cells for the test. In other words, the presence of leukemia cells with
the highest
MDR1 activity would be missed with the standard efflux assay because they were
poorly
loaded with dye. The present results show that the accumulation assay, which
reflects
accumulation which is independent of efflux (inhibited by zosuquidar),
frequently yielded
a much more sensitive assay for the assessment of leukemia cells with high
MDR1
activity. Furthermore, by loading such cells in the presence of an MDR1
inhibitor such as
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zosuquidar, an enhanced efflux assay was achieved (Figure 1 vs. Figure 4).
These novel
observations indicate that the use of the accumulation assay, or the efflux
assay with cells
loaded with dye in the presence of zosuquidar, are far superior methods over
the standard
efflux assay, for the assessment of MDRl status in patients with cancer.
All references cited herein, including but not limited to published and
unpublished
applications, patents, and literature references, are incorporated herein by
reference in
their entirety and are hereby made a part of this specification. To the extent
publications
and patents or patent applications incorporated by reference contradict the
disclosure
contained in the specification, the specification is intended to supersede
and/or take
precedence over any such contradictory material.
The term "comprising" as used herein is synonymous with "including,"
"containing," or "characterized by," and is inclusive or open-ended and does
not exclude
additional, unrecited elements or method steps.
All numbers expressing quantities of ingredients, reaction conditions, and so
forth
used in the specification are to be understood as being modified in all
instances by the
term "about." Accordingly, unless indicated to the contrary, the numerical
parameters set
forth herein are approximations that may vary depending upon the desired
properties
sought to be obtained. At the very least, and not as an attempt to limit the
application of
the doctrine of equivalents to the scope of any claims in any application
claiming priority
to the present application, each numerical parameter should be construed in
light of the
number of significant digits and ordinary rounding approaches.
The above description discloses several methods and materials of the present
invention. This invention is susceptible to modifications in the methods and
materials, as
well as alterations in the fabrication methods and equipment. Such
modifications will
become apparent to those skilled in the art from a consideration of this
disclosure or
practice of the invention disclosed herein. Consequently, it is not intended
that this
invention be limited to the specific embodiments disclosed herein, but that it
cover all
modifications and alternatives coming within the true scope and spirit of the
invention.
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