Note: Descriptions are shown in the official language in which they were submitted.
CA 02599884 2007-09-12
COMBINATIONS OF DRUGS (E.G., CIiLORPROMAZINE AND
s PENTAMIDINE) FOR THE TREATMENT OF NEOPLASTIC
DISORDERS
Background of the Invention
The invention relates to the treatment of neoplastic disorders such as
cancer.
Cancer is a disease marked by the uncontrolled growth of abnormal
cells. The abnormal cells may no longer do the work of normal cells, and they
crowd out and destroy healthy tissue.
Lung cancer is the most common cancer-related cause of death among
men and women. It is the second most commonly occurring cancer among
men and women; it has been estimated that there will be more than 164,000
new cases of lung cancer in the U.S. in the year 2000 alone. While the rate of
'ung cancer cases is declining among men in the U.S., it continues to increase
2o among women. Lung cancer can be lethal; according to the American Lung
Association, an estimated 156,900 Americans are expected to die due to lung
cancer in 2000.
Cancers that begin in the lungs are divided into two major types, non-
small cell lung cancer and small cell lung cancer, depending on how the cells
appear under a microscope. Non-small cell lung cancer (squamous cell
carcinoma, adenocarcinoma, and large cell carcinoma) generally spreads to
other organs more slowly than does small cell lung cancer. Small cell lung
cancer is the less common type, accounting for about 20% of all lung cancer.
CA 02599884 2007-09-12
Other cancers include brain cancer, breast cancer, cervical cancer, colon
cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma,
ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma,
skin
cancer, testicular cancer, and uterine cancer. These cancers, like lung
cancer,
are sometimes treated with chemotherapy.
Chemotherapeutic drugs currently in use or in clinical trials include
paclitaxel, docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin,
etoposide,
topotecan, irinotecan, anastrozole, rituximab, trastuzumab, fludarabine,
cyclophosphamide, gentuzumab, carboplatin, interferon, and doxorubicin. The
io most commonly used anticancer agent is paclitaxel, which is used alone or
in
combination with other chemotherapy drugs such as: 5-FU, doxorubicin,
vinorelbine, cytoxan, and cisplatin.
Summary of the Invention
We have discovered that the combination of the antipsychotic drug
chlorpromazine and the antiprotozoal drug pentamidine exhibits substantial
antiproliferative activity against cancer cells. Structural and functional
analogs
of each of these compounds are laaown, and any of these analogs can be used in
the antiproliferative combination of the invention. Metabolites of
chlorpromazine and pentamidine are also known. Many of these metabolites
share one or more biological activities with the parent compound and,
accordingly, can also be used in the antiproliferative combination of the
invention. Accordingly, the invention features a method for treating a patient
having a cancer or other neoplasm, by administering to the patient
chlorpromazine and pentamidine simultaneously or within 14 days of each
other in amounts sufficient to inhibit the growth of the neoplasm.
Preferably, the two compounds are administered within ten days of each
other, more preferably within five days of each other, and most preferably
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CA 02599884 2007-09-12
within twenty-four hours of each other or even simultaneously. The cancer
treated according to any of the methods of the invention, described below, can
be lung cancer (squamous cell carcinoma, adenocarcinoma, or large cell
carcinoma), brain cancer, breast cancer, cervical cancer, colon cancer,
gastric
cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer,
pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer,
testicular cancer, or uterine cancer.
In a related aspect, the invention also features a method for treating a
patient having a neoplasm such as cancer. In this method, the patient is
io administered (a) a first compound selected from prochlorperazine,
perphenazine, mepazine, methotrimeprazine, acepromazine, thiopropazate,
perazine, propiomazine, putaperazine, thiethylperazine, methopromazine,
chlorfenethazine, cyamemazine, perphenazine, enanthate, norchlorpromazine,
trifluoperazine, thioridazine (or a salt of any of the above), and dopamine D2
antagonists (e.g., sulpride, pimozide, spiperone, ethopropazine, clebopride,
bupropion, and haloperidol); and (b) a second compound selected from
propamidine, butamidine, heptamidine, nonamidine, stilbamidine,
hydroxystilbamidine, diminazene, benzamidine, 4,4'- (pentamethylenedioxy)
di-, dihydrochloride, phenamidine, dibrompropamidine, 1,3-bis(4-amidino-2-
methoxyphenoxy)propane, phenamidine, and amicarbalide (or a salt of any of
the above). Alternatively, the second compound can be a functional analog of
pentamidine, such as netropsin, distamycin, bleomycin, actinomycin, or
daunorubicin. The first and second compounds are preferably administered
simultaneously or within 14 days of each other and in amounts sufficient to
inhibit the growth of the neoplasm.
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CA 02599884 2007-09-12
In another related aspect, the invention also features a method for
treating a patient having a neoplasm such as cancer by administering the
following:
a) a first compound having the formula (I):
R, Rlo Ry
R2 I R8
9
3 I I 7
R 4 6
3 W R7
5 6
wherein R2 is selected from the group consisting of
--='=CF3 A-1 CH3
- CI A-2
F 0 A-7
A-3
-----OCH3 A-4
CH3
CH3 ~
~ \\O A-8
O A-5
-- -S
-N
A-6 CH3
A-9
-=--=OCP3 A-10
Rlo is selected from the group consisting of:
4
CA 02599884 2007-09-12
......
B-1 N B B-11
O
Q
'CH3
H H3
B-12
.....~N = NH2
3.2
N
H3 B-13
O ----- ",HCH3
H3
. B-14
B-3 B 8
J~ H~ H3 N-CH3 N-OH
H3G H3d'
B-4 ..--- B-15
~H3 ~H3
H3H3 H3~~H3 ~ H3
B-5 --
= B 16
B-10
CH3
.
N-CH3 NHOH
p OH
each of Rl, R3, R4, R5, R6, R7, R8, and R9 is, independently, H, F, OH, OCF3,
or
OCH3; and W is selected from the group consisting of:
5=o~~o S*
C-2 0 C-3
(Y.= N~.~ C==.
C-4 H C-5 H2 C-6
HC =CH
C-7
and
5
CA 02599884 2007-09-12
b) a second compound having the formula (II):
Ru
R13 I Y--(CHZ)n-Z P\\-I;)
R11 R12
wherein each of Y and Z is, independently, 0 or N; each of Rl l and R12 is,
independently, H, OH, Cl, Br, OCH3, OCF3, NO2, or NH2; n is an integer
between 2 and 6, inclusive; and each of R13 and R14 is, independently, at the
meta or para position and is selected from the group consisting of
----- NH ----- NOH ----- </// ND ND --- </// -
HaC
wherein the first and second compounds are administered simultaneously or
within 14 days of each other in amounts sufficient to inhibit the growth of
the
neoplasm.
In any of the foregoing treatments, each compound that is part of the
combination is preferably administered to the patient as part of a
pharmaceutical composition that also includes a pharmaceutically acceptable
carrier. Chlorpromazine is preferably administered at a dosage of 10 to 2500
milligrams and pentamidine is preferably administered at a dosage of 1 to 1000
milligrams. Preferred modes of administra.tion include intravenous,
intramuscular, inhalation, and oral administration.
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CA 02599884 2007-09-12
Various embodiments of this invention provide use of chlorpromazine and
pentamidine in
the treatment of a patient having a neoplasm to inhibit growth of the
neoplasm, wherein the
chlorpromazine and pentamidine are for administration simultaneously or within
fourteen days of
each other.
Other embodiments of this invention provide use of: (a) a first compound
selected from
the group consisting of prochlorperazine, perphenazine, mepazine,
methotrimeprazine,
acepromazine, thiopropazate, perazine, propiomazine, butaperazine,
thiethylperazine,
methopromazine, chlorfenethazine, cyamemazine, perphenazine, enanthate, and
norchlorpromazine, and (b) a second compound selected from the group
consisting of propamidine,
butamidine, heptamidine, nonamidine, stilbamidine, hydroxystilbamidine,
diminazene,
dibrompropamidine, 1,3-bis(4-amidino-2-methoxyphenoxy)propane, netropsin,
distamycin,
phenamidine, amicarbalide, and bleomycin, for the treatment of a patient
having a neoplasm to
inhibit growth of said neoplasm, wherein said first compound and said second
compound are for
administration simultaneously or within fourteen days of each other.
Other embodiments of this invention provide a pharmaceutical composition
comprising a
first compound, a second compound, and a pharmaceutically acceptable carrier,
wherein said first
compound is selected from the group consisting of prochlorperazine,
perphenazine, mepazine,
methotrimeprazine, acepromazine, thiopropazate, perazine, propiomazine,
butaperazine,
thiethylperazine, methopromazine, chlorfenethazine, cyamemazine, perphenazine,
enanthate, and
norchlorpromazine, and said second compound is selected from the group
consisting of
propamidine, butamidine, heptamidine, nonamidine, stilbamidine,
hydroxystilbamidine,
diminazene, dibrompropamidine, 1,3-bis(4-amidino-2-methoxyphenoxy)propane,
netropsin,
distamycin, phenamidine, amicarbalide, and bleomycin, wherein said first
compound and said
second compound are present in amounts that, when administered together to a
patient having a
neoplasm, reduce cell proliferation in said neoplasm.
Other embodiments of this invention provide a pharmaceutical pack comprising a
first
compound and a second compound, wherein said first compound is selected from
the group
consisting of prochlorperazine, perphenazine, mepazine, methotrimeprazine,
acepromazine,
thiopropazate, perazine, propiomazine, butaperazine, thiethylperazine,
methopromazine,
chlorfenethazine, cyamemazine, perphenazine, enanthate, and norchlorpromazine,
and said second
compound is selected from the group consisting of propamidine, butamidine,
heptamidine,
nonamidine, stilbamidine, hydroxystilbamidine, diminazene, dibrompropaniidine,
1,3-bis(4-
amidino-2-methoxyphenoxy)propane, netropsin, distamycin, phenamidine,
amicarbalide, and
bleomycin.
6a
CA 02599884 2007-09-12
The antiproliferative combinations of the invention can be part of a
pharmaceutical pack. Preferably, the chlorpromazine and pentamidine are
formulated together or separately and in individual dosage amounts.
It will be understood by those in the art that the compounds are also
useful when formulated as salts. For example, as is described herein, the
isethionate salt of pentamidine exhibits synergistic antiproliferative
activity
when combined with chlorpromazine. Other salts of pentamidine include the
platinum salt, the dihydrochloride salt, and the dimethanesulfonate salt (see,
for
example, Mongiardo et al., Lancet 2:108, 1989). Similarly, chlorpromazine
io salts include, for example, hydrochloride salts and maleate salts.
The invention also features methods for identifying compounds useful
for treating a patient having a neoplasm. The method includes the steps of
contacting cancers cell in vitro with (i) pentamidine or chlorpromazine and
(ii)
a candidate compound, and determining whether the cancer cells grow more
slowly than (a) cancer cells contacted with the chlorpromazine or pentamidine
but not contacted with the candidate compound, and (b) cancer cells contacted
with the candidate compound but not with chlorpromazine or pentamidine. A
candidate compound that, when combined with chlorpromazine or
pentamidine, reduces cell proliferation but, in the absence of chlorpromazine
or
pentamidine, does not is a compound that is useful for treating a patient
having
a neoplasm.
Combination therapy may be provided wherever chemotherapy is
performed: at home, the doctor's office, a clinic, a hospital's outpatient
department, or a hospital. Treatment generally begins at a hospital so that
the
doctor can observe the therapy's effects closely and make any adjustments that
are needed. The duration of the combination therapy depends on the kind of
cancer being treated, the age and condition of the patient, the stage and type
of
the patient's disease, and how the patient's body responds to the treatment.
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CA 02599884 2007-09-12
Drug administration may be performed at different intervals (e.g., daily,
weekly, or monthly) and the administration of each agent can be deterrnined
individually. Combination therapy may be given in on-and-off cycles that
include rest periods so that the patient's body has a chance to build healthy
new
cells and regain its strength.
Depending on the type of cancer and its stage of development, the
combination therapy can be used to treat cancer, to slow the spreading of the
cancer, to slow the cancer's growth, to kill or arrest cancer cells that may
have
spread to other parts of the body from the original tumor, to relieve symptoms
io caused by the cancer, or to prevent cancer in the first place. Combination
therapy can also help people live more comfortably by eliminating cancer cells
that cause pain or discomfort.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
Detailed Description of the Invention
We have discovered that the combination of the antipsychotic drug
chlorpromazine and the antiprotozoal drug pentamidine exhibits substantial
antiproliferative activity against cancer cells. Concentrations that exhibited
maximal antiproliferative activity against cancer cells were not toxic to
normal
cells. Thus, this drug combination is useful for the treatment of cancer and
other neoplasms.
Based on known properties that are shared between chlorpromazine and
its analogs and metabolites, and between pentamidine and its analogs and
metabolites, it is likely that structurally related compounds can be
substituted
for chlorpromazine and/or pentamidine in the antiproliferative combinations of
the invention. Information regarding each of the drugs and its analogs and
metabolites is provided below.
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Phenothiazines
Phenothiazines that are useful in the antiproliferative combination of the
invention are compounds having the general formula (I):
R, i lo Rs
R2 N Re
9
3 87
R 4 6
3 W RT
R4 Rs (I)
wherein R2 is selected from the group consisting of:
CH,
-----CF3 A-1
CH3
-----CI
A-2 -----
o A-7 -----S
-----F 0 A-5
A-3 CH3
------ =N CH3 A-9
-----OCH3
A-4 A-6
OCF3 A-10
0 A-g
io each of Rl, R3, R4, R5, R6, R7, R8, and R9 is, independently, H, OH, F,
OCF3,or
OCH3; W is selected from the group consisting of:
, S-
_S
C-l 0 0 C-2 II C-3
O, N,, ,C,
C-4 H C-5 HZ C-6
HC =CH
C-7
and Rlo is selected from the group consisting of:
9
CA 02599884 2007-09-12
----- . N
B-1 B-6 --- - B-11
~-N O
\CH3 ~
OH CH3
B-12
.....~
.... ~
Q B2 2
B-7
N-CH3 B-13
\OCH3 ~CH3
~N H ..... ~ ~ O B-8 B-14
B-3 N-CH3 H3 N-CH3 N OH
H3C~ H3C~
B-4 B-9
.,._1 ,CH3 ... ~H3 .._..~B-15
H3C/-N\CH3 H3~~-CH3 HN-CH3
. B-5 ===
B-10 B-16
CH3
N=~ NCH3 NHOH
p OH
In preferred compounds, R2 is Cl; each of Rl, R3, R4, R5, R6, R7, R8, and R9
is H
or F. More preferably, each of Rl, R4, R5, R6, and R9 is H.
The most commonly prescribed member of the phenothiazine family is
chiorpromazine, which has the structure:
CA 02599884 2007-09-12
1H3
N
~-ICH3
CI N
B-1
Chlorpromazine is currently available in the following forms: tablets,
capsules,
suppositories, oral concentrates and syrups, and formulations for injection.
Phenothiazines considered to be chlorpromazine analogs include
fluphenazine, prochlorperazine, thioridazine, and trifluoperazine. Many of
these share antipsychotic or antiemetic activity with chlorpromazine.
Phenothiazines are thought to elicit their antipsychotic and antiemetic
effects via interference with central dopaminergic pathways in the mesolimbic
io and medullary chemoreceptor trigger zone areas of the brain. Extrapyramidal
side effects are a result of interactions with dopaminergic pathways in the
basal
ganglia. Although often termed dopamine blockers, the exact mechanism of
dopaminergic interference responsible for the drugs' antipsychotic activity
has
not been determined. Chlorpromazine has strong alpha-adrenergic blocking
activity and can cause orthostatic hypotension. Chlorpromazine also has
moderate anticholinergic activity manifested as occasional dry mouth, blurred
vision, urinary retention, and constipation. Chlorpromazine increases
prolactin
secretion owing to its dopamine receptor blocking action in the pituitary and
hypothalamus.
Chlorpromazine is readily absorbed from the gastrointestinal tract. Its
bioavailability is variable due to considerable first pass metabolism by the
liver. Liquid concentrates may have greater bioavailability than tablets. Food
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CA 02599884 2007-09-12
does not appear to affect bioavailability consistently. I.m. administration
bypasses much of the first pass effect and higher plasma concentrations are
achieved. The onset of action after i.m. administration is usually 15 to 30
minutes and after oral administration 30 to 60 minutes. Rectally administered
s chlorpromazine usually takes longer to act than orally administered
chlorpromazine.
Clzlorpromazine Metabolites
Because chlorpromazine undergoes extensive metabolic transformation
to into a number of metabolites that may be therapeutically active, these
metabolites may be substituted from chlorpromazine in the antiproliferative
combination of the invention. The metabolism of chlorpromazine yields, for
example, oxidative N-demethylation to yield the corresponding primary and
secondary amine, aromatic oxidation to yield a phenol, N-oxidation to yield
the
15 N-oxide, S-oxidation to yield the sulphoxide or sulphone, oxidative
deamination of the aminopropyl side chain to yield the phenothiazine nuclei,
and glucuronidation of the phenolic hydroxy groups and tertiary amino group
to yield a quatemary ammonium glucuronide.
In other examples of chlorpromazine metabolites useful in the
antiproliferative
20 combination of the invention, each of positions 3, 7, and 8 of the
phenothiazine
can independently be substituted with a hydroxyl or methoxyl moiety.
Pentamidine
Pentamidine is currently used for the treatment of Pneuinocystis carinii,
25 Leishrnania donovani, Trypanosoma brucei, T. gambiense, and T. rhodesiense
infections. The structure of pentamidine is:
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CA 02599884 2007-09-12
NH NH
H2N \ I \ I NFiz
= F-1
It is available formulated for injection or inhalation. For injection,
pentamidine
is packaged as a nonpyrogenic, lyophilized product. After reconstitution, it
is
administered by intramuscular or intravenous injection.
Pentamidine isethionate is a white, crystalline powder soluble in water
and glycerin and insoluble in ether, acetone, and chloroform. It is chemically
designated 4,4'- diamidino-diphenoxypentane di(P-hydroxyethanesulfonate).
The molecular formula is C23H36N4010S2 and the molecular weight is 592.68.
The mode of action of pentamidine is not fully understood. In vitro
studies with mammalian tissues and the protozoan Crithidia oncopelti indicate
that the drug interferes with nuclear metabolism, producing inhibition of the
synthesis of DNA, RNA, phospholipids, and proteins.
Little is also known about the drug's pharmacokinetics. In seven
patients treated with daily i.m. doses of pentamidine at 4 mg/kg for 10 to 12
days, plasma concentrations were between 0.3 and 0.5 g/mL. The patients
continued to excrete decreasing amounts of pentamidine in urine up to six to
eight weeks after cessation of the treatment.
Tissue distribution of pentamidine has been studied in mice given a
single intraperitoneal injection of pentamidine at 10 mg/kg. The concentration
in the kidneys was the highest, followed by that in the liver. In mice,
pentamidine was excreted unchanged, primarily via the kidneys with some
elimination in the feces. The ratio of amounts excreted in the urine and feces
(4:1) was constant over the period of study.
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Pentanzidine Analogs
Aromatic diamidino compounds can replace pentamidine in the
antiproliferative combination of the invention. Aromatic diamidino
compounds such as propamidine, butamidine, heptamidine, and nonamidine
share properties with pentamidine in that they exhibit antipathogenic or DNA
binding properties. Other analogs (e.g., stilbamidine and indole analogs of
stilbamidine, hydroxystilbamidine, diminazene, benzamidine, 4,4-
(pentamethylenedioxy) di-, dihydrochloride, phenamidine, dibrompropamidine,
1,3-bis(4-amidino-2-methoxyphenoxy)propane (DAMP), netropsin,
lo distamycin, phenamidine, amicarbalide, bleomycin, actinomycin, and
daunorubicin) also exhibit properties similar to those of pentamidine. It is
likely that these compounds will have antiproliferative activity when
administered in combination with chlorpromazine (or an analog or metabolite
of chlorpromazine).
Preferred analogs are described, for example, by formula (II).
~
/1a
Y-(CH2)n'Z \ /
R13
RII R12 !TT\
wherein each of Y and Z is, independently, 0 or N; each of Rl l and R12 is,
independently, H, OH, Cl, Br, OCH3, OCF3, NOZ, or NH2; and n is an integer
from 2 to 6, inclusive; and each of R13 and R14 is, independently, at the meta
or
para position and is selected from the group consisting of:
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CA 02599884 2007-09-12
NH NOH N N N\
----- ----- -----~ ----- / ----- / II
~
D-1 NHz D-2 NH2 D-3 N D-4 N D-5 N/-N
H H
H3C
Other analogs include stilbamidine (G-1) and hydroxystilbamidine (G-
2), and their indole analogs (e.g., G-3).
HZN
\ / \ NH
HN
NH2
G-1
OH
H2N / \
\ / \ NH
HN -
NH2
G-2
NH
HZN N NHz
HN
G-3
Each amidine moiety may be replaced with one of the moieties depicted as D-
2, D-3, D-4, or D-5, above. As is the case for the phenothiazines and
pentamidine, salts of stilbamidine and its related compounds are also useful
in
io the method of the invention. Preferred salts include, for example,
dihydrochloride and methanesulfonate salts.
Pentanaidine Metabolites
Pentamidine metabolites are also useful in the antiproliferative
combination of the invention. Pentamidine is rapidly metabolized in the body
to at least seven primary metabolites. Some of these metabolites share one or
CA 02599884 2007-09-12
more activities with pentamidine. It is likely that some pentamidine
metabolites will exhibit antiproliferative activity when combined with
chlorpronlazine or an analog thereof.
Seven pentamidine metabolites are shown below.
HN HN
(CH2)d COOH ~ \ O(C}i2)aCHpOH
HZN - G 1 H2N - ~
HN NH NOH
OH
HZN G_z H2N \ I \ I NHp
O O {
G'7
NH NH
N
Hp
HZN ~ I e
O O
H G-3
NH NH
HZN \ ~ OH \ I NHZ
0
G-4
NOH NOH
H2N \ ( \ I NHZ
G-S
Thera
The combinations of compounds of the invention are useful for the
treatment of neoplasms. Combination therapy may be performed alone or in
conjunction with another therapy (e.g., surgery, radiation, chemotherapy,
biologic therapy). Additionally, a person having a greater risk of developing
a
16
CA 02599884 2007-09-12
neoplasm (e.g., one who is genetically predisposed or one who previously had
a neoplasm) may receive prophylactic treatment to inhibit or delay neoplastic
formation.
The administration dosage and frequency of each component can be
controlled independently. For example, one compound may be administered
orally three times per day, while the second compound may be administered
intramuscularly once per day. The compounds may also be formulated
together such that one administration delivers both components. Formulations
and dosages are described below.
Formulation of Pharmaceutical Compositions
The administration of each compound of the combination may be by any
suitable means that results in a concentration of the compound that, combined
with the other component, is specifically anti-neoplastic upon reaching the
target region. The compound may be contained in any appropriate amount in
any suitable carrier substance, and is generally present in an amount of 1-95%
by weight of the total weight of the composition. The composition may be
provided in a dosage form that is suitable for the oral, parenteral (e.g.,
intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal, inhalent,
skin
(patch), or ocular administration route. Thus, the composition may be in form
of, e.g., tablets, capsules, pills, powders, granulates, suspensions,
emulsions,
solutions, gels including hydrogels, pastes, ointments, creams, plasters,
drenches, delivery devices, suppositories, enemas, injectables, implants,
sprays,
or aerosols. The pharmaceutical compositions may be formulated according to
conventional pharmaceutical practice (see, e.g., Remington: The Science and
Practice of Pharmacy, (19th ed.) ed. A.R. Gennaro, 1995, Mack Publishing
Company, Easton, PA. and Encyclopedia of Pharmaceutical Technology, eds.
J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.
17
CA 02599884 2007-09-12
Pharmaceutical compositions according to the invention may be
formulated to release the active compound substantially immediately upon
administration or at any predetermined time or time period after
administration.
The latter types of compositions are generally known as controlled release
formulations, which include (i) formulations that create a substantially
constant
concentration of the drug within the body over an extended period of time;
(ii)
fomiulations that after a predetennined lag time create a substantially
constant
concentration of the drug within the body over an extended period of time;
(iii)
formulations that sustain drug action during a predetermined time period by
maintaining a relatively, constant, effective drug level in the body with
concomitant minimization of undesirable side effects associated with
fluctuations in the plasma level of the active drug substance (sawtooth
kinetic
pattern); (iv) formulations that localize drug action by, e.g., spatial
placement
of a controlled release composition adjacent to or in the diseased tissue or
organ; and (v) formulations that target drug action by using carriers or
chemical
derivatives to deliver the drug to a particular target cell type.
Administration of compounds in the form of a controlled release
formulation is especially preferred in cases in which the compound, either
alone or in combination, has (i) a narrow therapeutic index (i.e., the
difference
2o between the plasma concentration leading to harmful side effects or toxic
reactions and the plasma concentration leading to a therapeutic effect is
small;
in general, the therapeutive index, TI, is defined as the ratio of median
lethal
dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption window
in the gastro-intestinal tract; or (iii) a very short biological half-life so
that
frequent dosing during a day is required in order to sustain the plasma level
at a
therapeutic level.
Any of a number of strategies can be applied in order to obtain a
controlled release formulation in which the rate of release outweighs the rate
of
18
CA 02599884 2007-09-12
metabolism of the compound in question. In one example, controlled release is
obtained by appropriate selection of various formulation parameters and
ingredients, including, e.g., various types of controlled release compositions
and coatings. Thus, the drug substance is formulated with appropriate
excipients into a pharmaceutical composition that, upon administration to the
organism, releases the active substance in a controlled manner. Examples
include single or multiple unit tablet or capsule compositions, oil solutions,
suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches,
and liposomes.
Solid Dosage Forn:s For Oral Use
Formulations for oral use include tablets containing the active
ingredient(s) in a mixture with non-toxic pharmaceutically acceptable
excipients. These excipients may be, for example, inert diluents or fillers
(e.g.,
sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches
including
potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate,
calcium sulfate, or sodium phosphate); granulating and disi.ntegrating agents
(e.g., cellulose derivatives including microcrystalline cellulose, starches
including potato starch, croscarmellose sodium, alginates, or alginic acid);
2o binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,
sodium
alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose,
magnesium aluminum silicate, carboxymethylcellulose sodium,
methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,
polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,
glidants,
and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,
silicas,
hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable
excipients can be colorants, flavoring agents, plasticizers, humectants,
buffering agents, and the like.
19
CA 02599884 2007-09-12
The tablets may be uncoated or they may be coated by known
techniques, optionally to delay disintegration and absorption in the
gastrointestinal tract and thereby providing a sustained action over a longer
period. The coating may be adapted to release the active drug substance in a
predetermined pattern (e.g., in order to achieve a controlled release
formulation) or it may be adapted not to release the active drug substance
until
after passage of the stomach (enteric coating). The coating may be a sugar
coating, a film coating (e.g., based on hydroxypropyl methylcellulose,
methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose,
io carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or
polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid
copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose
phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate
phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material
such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
The solid tablet compositions may include a coating adapted to protect
the composition from unwanted chemical changes, (e.g., chemical degradation
prior to the release of the active drug substance). The coating may be applied
on the solid dosage form in a similar manner as that described in Encyclopedia
of Pharrnaceutical Technolouy, supra.
The two drugs may be mixed together in the tablet, or may be
partitioned. In one example, the first drug is contained on the inside of the
tablet, and the second drug is on the outside, such that a substantial portion
of
the second drug is released prior to the release of the first drug.
Formulations for oral use may also be presented as chewing tablets, or
as hard gelatin capsules wherein the active ingredient is mixed with an inert
solid diluent (e.g., potato starch, lactose, microcrystalline cellulose,
calcium
carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein
the
CA 02599884 2007-09-12
active ingredient is mixed with water or an oil medium, for example, peanut
oil, liquid paraffin, or olive oil. Powders and granulates may be prepared
using
the ingredients mentioned above under tablets and capsules in a conventional
manner using, e.g., a mixer, a fluid bed apparatus or a spray drying
equipment.
Controlled Release Oral Dosage Forins
Controlled release compositions for oral use may, e.g., be constructed to
release the active drug substance by controlling the dissolution and/or the
1o diffusion of the active drug substance.
Dissolution or diffusion controlled release can be achieved by
appropriate coating of a tablet, capsule, pellet, or granulate formulation of
compounds, or by incorporating the compound in question into an appropriate
matrix.
A controlled release coating may include one or more of the coating substances
mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax,
carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate,
glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid,
cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl
pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-
hydroxymethacrylate, methacrylate hydrogels, 1,3butylene glycol, ethylene
glycol methacrylate, and/or polyethylene glycols. In a controlled release
matrix formulation, the matrix material may also include, e.g., hydrated
metylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone,
glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride,
polyethylene, and/or halogenated fluorocarbon.
A controlled release composition containing one or more of the
compounds of the claimed combinations may also be in the form of a buoyant
21
CA 02599884 2007-09-12
tablet or capsule (i.e., a tablet or capsule that, upon oral administration,
floats
on top of the gastric content for a certain period of time. A buoyant tablet
formulation of the compound(s) can be prepared by granulating a mixture of
the drug(s) with excipients and 20-75% w/w of hydrocolloids, such as
hydroxyethrlcellulose, hydroxypropylcellulose and
hydroxypropylmethylcellulose. The obtained granules can then be compressed
into tablets. On contact with the gastric juice, the tablet forms a
substantially
water-impermeable gel barrier around its surface. This gel barrier takes part
in
maintaining a density of less than one, thereby allowing the tablet to remain
lo buoyant in the gastric juice.
Liquids for Oral Adininistration
Powders, dispersible powders, or granules suitable for preparation of an
aqueous suspension by addition of water are convenient dosage forms for oral
administration. Formulation as a suspension provides the active ingredient in
a
mixture with a dispersing or wetting agent, suspending agent, and one or more
preservatives. Suitable dispersing or wetting agents are, for example,
naturally-occurring phosphatides (e.g., lecithin or condensation products of
ethylene oxide with a fatty acid, a long chain aliphatic alcohol, or a partial
ester
2o derived from fatty acids) and a hexitol or a hexitol anhydride (e.g.,
polyoxyethylene stearate, polyoxyethylene sorbitol monooleate,
polyoxyethylene sorbitan monooleate, and the like). Suitable suspending
agents are, for example, sodium carboxymethylcellulose, methylcellulose,
sodium alginate, and the like.
Parenteral Con:positions
The pharmaceutical composition may also be administered parenterally
by injection, infusion or implantation (intravenous, intramuscular,
22
CA 02599884 2007-09-12
subcutaneous, or the like) in dosage forms, formulations, or via suitable
delivery devices or implants containing conventional, non-toxic
pharmaceutically acceptable carriers and adjuvants. The formulation and
preparation of such compositions is well-lmown to those skilled in the art of
pharmaceutical formulation. Specific formulations can be found in Remington:
The Science and Practice of Pharniacy, supra.
Compositions for parenteral use may be presented in unit dosage forms
(e.g., in single-dose ampoules), or in vials containing several doses and in
which a suitable preservative may be added (see below). The composition may
io be in form of a solution, a suspension, an emulsion, an infusion device, or
a
delivery device for implantation, or it may be presented as a dry powder to be
reconstituted with water or another suitable vehicle before use. Apart from
the
active drug(s), the composition may include suitable parenterally acceptable
carriers and/or excipients. The active drug(s) may be incorporated into
microspheres, microcapsules, nanoparticles, liposomes, or the like for
controlled release. Furthermore, the composition may include suspending,
solubilizing, stabilizing, pH-adjusting agents, and/or dispersing agents.
As indicated above, the pharmaceutical compositions according to the
invention may be in the form suitable for sterile injection. To prepare such a
composition, the suitable active drug(s) are dissolved or suspended in a
parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents
that may be employed are water, water adjusted to a suitable pH by addition of
an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable
buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride
solution. The aqueous formulation may also contain one or more preservatives
(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the
23
CA 02599884 2007-09-12
compounds is only sparingly or slightly soluble in water, a dissolution
enhancing or solubilizing agent can be added, or the solvent may include 10-
60% wlw of propylene glycol or the like.
Controlled Release Parenteral Compositions
Controlled release parenteral compositions may be in form of aqueous
suspensions, microspheres, microcapsules, magnetic microspheres, oil
solutions, oil suspensions, or emulsions. Alternatively, the active drug(s)
may
be incorporated in biocompatible carriers, liposomes, nanoparticles, implants,
lo or infusion devices.
Materials for use in the preparation of microspheres and/or
microcapsules are, e.g., biodegradable/bioerodible polymers such as
polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamnine)
and, poly(lactic acid).
Biocompatible carriers that may be used when formulating a controlled release
parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g.,
albumin), lipoproteins, or antibodies.
Materials for use in implants can be non-biodegradable (e.g.,
polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic
2o acid), poly(glycolic acid) or poly(ortho esters)).
Rectal Compositions
For rectal application, suitable dosage forms for a composition include
suppositories (emulsion or suspension type), and rectal gelatin capsules
(solutions or suspensions). In a typical suppository formulation, the active
drug(s) are combined with an appropriate pharmaceutically acceptable
suppository base such as cocoa butter, esterified fatty acids, glycerinated
24
CA 02599884 2007-09-12
gelatin, and various water-soluble or dispersible bases like polyethylene
glycols
and polvoxyethylene sorbitan fatty acid esters. Various additives, enhancers,
or surfactants may be incorporated.
Conipositions for Inl:alation
For administration by inhalation, typical dosage forms include nasal
sprays and aerosols. In a typically nasal formulation, the active
ingredient(s)
are dissolved or dispersed in a suitable vehicle. The pharmaceutically
acceptable vehicles and excipients (as well as other pharmaceutically
lo acceptable materials present in the composition such as diluents,
enhancers,
flavoring agents, and preservatives) are selected in accordance with
conventional pharmaceutical practice in a manner understood by the persons
skilled in the art of formulating pharmaceuticals.
Percutaneous and Topical Compositions
The pharmaceutical compositions may also be administered topically on
the slcin for percutaneous absorption in dosage forms or formulations
containing conventionally non-toxic pharmaceutical acceptable carriers and
excipients including microspheres and liposomes. The formulations include
creams, ointments, lotions, liniments, gels, hydrogels, solutions,
suspensions,
sticks, sprays, pastes, plasters, and other kinds of transdermal drug delivery
systems. The pharmaceutically acceptable carriers or excipients may include
emulsifying agents, antioxidants, buffering agents, preservatives, humectants,
penetration enhancers, chelating agents, gelforming agents, ointment bases,
perfumes, and skin protective agents.
Examples of emulsifying agents are naturally occurring gums (e.g., gum
acacia or gum tragacanth) and naturally occurring phosphatides (e.g., soybean
lecithin and sorbitan monooleate derivatives). Examples of antioxidants are
CA 02599884 2007-09-12
butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof,
tocopherol and derivatives thereof, butylated hydroxy anisole, and cysteine.
Examples of preservatives are parabens, such as methyl or propyl p-
hydroxybenzoate, and benzalkonium chloride. Examples of humectants are
glycerin, propylene glycol, sorbitol, and urea. Examples of penetration
enhancers are propylene glycol, DMSO, triethanolamine, N,N-
dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and derivatives
thereof, tetrahydrofurfuryl alcohol, and AzoneT""® Examples of chelating
agents are sodium EDTA, citric acid, and phosphoric acid. Examples of gel
io forrning agents are CarbopolT"', cellulose derivatives, bentonite,
alginates, gelatin
and polyvinylpyrrolidone. Exarnples of ointment bases are beeswax, paraffin,
cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span),
polyethylene glycols, and condensation products between sorbitan esters of
fatty acids and ethylene oxide (e.g., polyoxyethylene sorbitan monooleate
(TweenTM)).
The pharniaceutical compositions described above for topical
administration on the skin may also be used in connection with topical
administration onto or close to the part of the body that is to be treated.
The
compositions may be adapted for direct application or for introduction into
relevant orifice(s) of the body (e.g., rectal, urethral, vaginal or oral
orifices).
The composition may be applied by means of special drug delivery devices
such as dressings or alternatively plasters, pads, sponges, strips, or other
forms
of suitable flexible material.
Controlled Release Percutaneous and Topical Compositions
There are several approaches for providing rate control over the release
and transdermal permeation of a drug, including: membrane-moderated
systems, adhesive diffusion-controlled systems, matrix dispersion-type
26
CA 02599884 2007-09-12
systems, and microreservoir systems. A controlled release percutaneous and/or
topical composition may be obtained by using a suitable mixture of the above-
mentioned approaches.
In a membrane-moderated system, the active drug is present in a
reservoir which is totally encapsulated in a shallow compartment molded from
a drug-impermeable laminate, such as a metallic plastic laminate, and a rate-
controlling polymeric membrane such as a microporous or a non-porous
polymeric membrane (e.g., ethylene-vinyl acetate copolymer). The active
compound is only permitted to be released through the rate-controlling
io polymeric membrane. In the drug reservoir, the active drug substance may
either be dispersed in a solid polymer matrix or suspended in a viscous liquid
medium such as silicone fluid. On the external surface of the polymeric
membrane, a thin layer of an adhesive polymer is applied to achieve an
intimate contact of the transdermal system with the skin surface. The adhesive
polymer is preferably a hypoallergenic polymer that is compatible with the
active drug.
In an adhesive diffusion-controlled system, a reservoir of the active drug
is formed by directly dispersing the active drug in an adhesive polymer and
then spreading the adhesive containing the active drug onto a flat sheet of
substantially drug-impermeable metallic plastic backing to form a thin drug
reservoir layer. A matrix dispersion-type system is characterized in that a
reservoir of the active drug substance is formed by substantially
homogeneously dispersing the active drug substance in a hydrophilic or
lipophilic polymer matrix and then molding the drug-containing polymer into a
2s disc with a substantially well-defined surface area and thickness. The
adhesive
polymer is spread along the circumference to form a strip of adhesive around
the disc.
27
CA 02599884 2007-09-12
In a microreservoir system, the reservoir of the active substance is
formed by first suspending the drug solids in an aqueous solution of water-
soluble polymer, and then dispersing the drug suspension in a lipophilic
polymer to form a plurality of microscopic spheres of drug reservoirs.
Dosaaes
The dosage of each compound of the claimed combination depends on
several factors, including: the administration method, the disease to be
treated,
the severity of the disease, whether the disease is to be treated or
prevented,
to and the age, weight, and health of the person to be treated.
The compounds are preferably administered in an amount of about 0.1-
30 mg/kg body weight per day, and more preferably in an amount of about 0.5-
mg/kg body weight per day. As described above, the compound in question
may be administered orally in the form of tablets, capsules, elixirs or
syrups, or
15 rectally in the form of suppositories. Parenteral administration of a
compound
is suitably performed in the form of saline solutions or with the compound
incorporated into liposomes. In cases where the compound in itself is not
sufficiently soluble to be dissolved, a solubilizer such as ethanol can be
applied. Below, for illustrative purposes, the dosages for chlorpromazine and
pentamidine are described. One in the art will recognize that if a second
compound is substituted for either chlorpromazine or pentamidine, the correct
dosage can be determined by examining the efficacy of the compound in cell
proliferation assays, as well as its toxicity in humans.
Oral Administration
For chlorpromazine adapted for oral administration for systemic use, the
dosage is normally about 1 mg to '1000 mg per dose administered (preferably
about 5 mg to 500 mg, and more preferably about 10 mg to 300 mg) one to ten
28
CA 02599884 2007-09-12
times daily (preferably one to 5 times daily) for one day to one year, and may
even be for the life of the patient; because the combinations of the invention
function primarily as cytostatic rather than cytotoxic agents, and exhibit low
toxicity, chronic, long-term administration will be indicated in many cases.
s Dosages up to 8 g per day may be necessary.
For pentamidine, the dosage is normally about 0.1 mg to 300 mg per
dose administered (preferably about 1 mg to 100 mg) one to four times daily
for one day to one year, and, like chlorpromazine, may be administered for the
life of the patient. Administration may also be given in cycles, such that
there
io are periods during which time pentamidine is not administered. This period
could be, for example, about a day, a week, a month, or a year or more.
Rectal Administration
For compositions adapted for rectal use for preventing disease, a
is somewhat higher amount of a compound is usually preferred. Thus a dosage of
chlorpromazine is normally about 5 mg to 2000 mg per dose (preferably about
mg to 1000 mg, more preferably about 25 mg to 500 mg) administered one
to four times daily. Treatment lengths are as described for oral
admininstration. The dosage of pentamidine is as described for orally
admininstered pentamidine.
Parenteral Administration
For intravenous or intramuscular administration of chlorpromazine, a
dose of about 0.1 mg/kg to about 100 mg/kg body weight per day is
2s recomnrnended, a dose of about 1 mg/kg to about 25 mg/kg is preferred, and
a
dose of 1 mg/kg to 10 mg/kg is most preferred. Pentamidine is administered at
29
CA 02599884 2007-09-12
a dose of about 0.1 mg/kg to about 20 mg/kg, preferably at a dose of about 0.5
mg/kg to about 10 mg/kg, and more preferably at a dose of about 1 mg/kg to
about 4 mg/kg.
Each compound is usually administered daily for up to about 6 to 12
months or more. It may be desirable to administer a compound over a one to
three hour period; this period may be extended to last 24 hours or more. As is
described for oral administration, there may be periods of about one day to
one
year or longer during which at least one of the drugs is not administered.
Inhadation
For inhalation, chlorpromazine is administered at a dose of about 1 mg
to 1000 mg daily, and preferably at a dose of about 10 mg to 500 mg daily. For
pentamidine, a dose of about 10 mg to 1000 mg, and preferably at a dose of 30
mg to 600 mg, is administered daily.
Percutaneous Administration
For topical administra.tion of either compound, a dose of about 1 mg to
about 5 g administered one to ten times daily for one week to 12 months is
usually preferable.
The following examples are to illustrate the invention. They are not
meant to limit the invention in any way.
Example 1: Preaaration of the chloraromazine/nentamidine isethionate
dilution matrix
Stock solutions of chlorpromazine and pentamidine isethionate (Sigma
catalog number C8138 and P0547, respectively) were made in
dimethylsulfoxide (DMSO) at concentrations of 11.25 mM and 6.74 mM
respectively. An 8X stock solution (128 M) of each individual compound
CA 02599884 2007-09-12
was made in Dulbecco's Modified Eagle Medium (DMEM) (Gibco 11995-040T"')
containing 10% fetal bovine serum (FBS), 200 m~l~i L-glutamine, and 1%
antibiotic/antimycotic solution. From this a 2-fold dilution series was made
in
DMEM. This series provided 9 concentrations ranging from 64 M to 240 nM
and one concentration of 0 M. The compound mixture matrix was prepared by
filling columns of a 384-well plate with the dilution series of
chlorprornazine
(first column: 32 M; second column: 16 .M; third column: 8 pM; fourth
column: 4 M; fifth column: 2 M; sixth column: 1 pM; seventh column: 500
nM; eighth column: 250 nM; ninth column: 125 nM; and tenth column: no
io compound) and filling the rows with the dilution series of pentaYnidine
(first
column: 32 pM; second column: 16 pM; third column: 8 pM; fourth column: 4
M; fifth column: 2 pM; sixth column: 1 M; seventh column: 500 nM; eighth
column: 250 nM; ninth column: 125 nM; and tenth column: no compound)
using a 16-channel pipettor (FinnpipetteT""). This compound mixture plate
i5 provided 4X concentrations of each compound that are transferred to assay
plates. The dilution matrix thus contained 100 different points - 81 wells
where varying amounts of chlorpromazine and pentamidine were present, as
well as a 10 point dilution series (2-fold) for each individual compound.
2o Examnle 2: Assay for Antioroliferative Activitv
The compound dilution matrix was assayed using the A549
bromodeoxyuridine (BrdU) cytoblot method. Forty-five microliters of a
suspension containing A5491ung adenocarcinoma cells (ATCC# CCL-185)
was seeded in a white opaque polystyrene cell culture treated sterile 384-well
25 plate (NalgeNunc #164610) using a multidrop (Labsystems) to give a density
of 3000 cells per well. Fifteen microliters of the 4X compound mixture matrix
was added to each well of the plate containing the cells. The compound
31
CA 02599884 2007-09-12
mixture matrix was transferred using a 16-channel pipettor (Finnpipette). In
addition, control wells with paclitaxel (final concentration 4.6 M),
podophyllotoxin (9.6 pM), and quinacrine (8.5 1VI) were added to each plate.
Each experiment was conducted in triplicate plates.
After incubation for 48 hours at 37 C, BrdU was added to each well at a
concentration of 10 pM. After 16 hours, the media was aspirated and the cells
were fixed by the addition of 70% ethanol and phosphate-buffered saline (PBS)
at room temperature for 1 hour. The fixative was aspirated and 2N HCl with
Tween 20 (polyoxyethylene sorbitan monolaurate) was added to each well and
lo the plates were incubated for 20 minutes at room temperature. The HCl was
neutcalized with a solution of 2N NaOH and the cells were washed twice with
Hank's Balanced Salt Solution (HBSS) and once with PBS containing 0.5%
bovine serum albumin (BSA) and 0.1% Tween 20. The wash solution was
removed and mouse anti-BrdU primary antibody (Pha.rMingenTM#555627) was
is diluted 1:1000 in PBS containing BSA, Tween 20, and secondary antibody at a
dilution of 1:2000 (AmershamTM #NA93 1). The secondary antibody recognizes
the mouse antibody and it is conjugated to the enzyme horseradish peroxidase
(HRP). After one hour of incubation, the antibody solution was removed and
the cells washed once with PBS. After the PBS wash, the HRP substrate
20 (which contains luminol, hydrogen peroxide, and an enhancer such as para-
iodophenol) was added to each well. The plates were read using an LJL
Analyst. All aspirations as well as the washes with PBS and HBSS were
performed using a TECAN Power Washer 384. The amount of light output
from each well indicates the amount of DNA synthesis that occurred in that
25 well. Decreased light indicates antiproliferative action of the compounds.
Luminescence for each position in the chlorpromazine/pentamidine
dilution matrix was divided into the luminescence values for A549 cells
treated
32
. CA 02599884 2007-09-12
with only DMSO vehicle, providing antiproliferative ratios for each position
in
the chlorpromazine/pentamidine dilution matrix. Antiproliferative ratios were
also calculated for paclitaxel, podophyllotoxin and quinacrine and used for
comparison.
Table--Antip=oliferative Ratios
Pentamidine Concentrations ( M)
8 4 2 1 0.5 0.25 0.13 0.06 0.03 0
8 6.8 6.3 5.1 3.7 2.7 3.1 2.4 2.3 2.3 2.6
4 7.8 9.7 5.9 5.5 4.0 4.3 4.1 3.2 2.8 2.9
2 8.4 9.0 4.8 4.5 5.0 3.6 3.2 3.0 2.3 2.4
1 8.0 6.9 5.1 5.6 4.7 3.3 2.8 2.3 1.9 1.8
0.5 7.6 7.0 4.4 4.9 2.9 3.3 1.9 1.8 1.6 1.7
U 0.25 6.6 6.1 3.7 6.2 3.7 3.8 1.6 1.6 1.3 1.3
a 0.13 6.2 4.7 3.0 5.2 2.8 2.4 1.4 1.3 1.1 1.2
. 0.06 5.0 5.0 3.1 3.0 2.3 2.0 1.3 1.1 1.2 1.0
0.03 4.3 4.1 3.2 3.0 2.6 1.9 1.2 1.1 1.0 1.1
a 0 4.9 3.9 3.0 2.9 3.0 2.3 1.4 1.2 0.9 1.0
0
a
U
At 4.0 M, pentamidine isethionate alone yields an antiproliferative ratio
of 3.9 and this increases to 4.9 when the concentration is doubled to 8.0 M.
Four micromolar chlorpromazine yields a ratio of 2.9, and this is increased no
further by doubling the concentration to 8.0 M. When 4.0 M pentamidine is
tested in combination with 4.0 M chlorpromazine (8.0 M total compound
species), an antiproliferative ratio of 9.7 is achieved.
In another analysis, the potency of the single compounds is shifted by
the presence of the other compound. The maximal antiproliferative ratio
achieved by pentamidine alone was 4.9 (at 8.0 M) and this was observed
when 1 M pentamidine was combined with chlorpromazine at concentrations
2o as low as 125 nM, significantly reducing the total drug species needed to
achieve this effect.
33
= = CA 02599884 2007-09-12
We demonstrated that the combination of chlorpromazine and
pentamidine isethionate arrests the growth of A549 cells without killing them.
A549 cells were seeded at subconfluence in 6-well plates and treated with 4
M of each drug for 72 hours. The medium was exchanged and the cells
cultured for seven days (with one medium change), at which time the cells
were counted and viability determined. Whereas untreated cells had grown to
confluence after seven days, the cells treated with chlorpromazine/pentamidine
isethionate had not grown, but had remained near the density at which they
were seeded. Additionally, 95% of chlorpromazine/pentamidine isethionate-
io treated cells were still viable, only slightly less than that observed for
the
untreated cells.
The combinations have little or no effect on the viability of normal lung
fibroblasts (MRC9). The control experiments demonstrating this are described
below. MRC9 cells were seeded into 3 84 well plates to confluence. A
chlorpromazine/pentamidine isethionate dilution matrix was added to give
overlapping 2-fold dilution series in a 12 X 12 matrix, with 16 M of each
compound being the maximal dose. The cells were incubated with the
compounds for 48 hours, after which time 6 L of Alamar Blue viability dye
was added to each well. The cells were incubated an additional 4 hours with
the dye. The amount of reduced dye was then determined by fluorescence
using an Analyst AD. The nonfluorescent (oxidized) form of the dye is
reduced to a fluorescent form by living cells. Thus, the ratio of fluorescence
between the wells that did not receive compounds over those that did receive
compounds is proportional to the amount of cell death caused by the
compounds. In this experiment, no toxic effects were present until each drug
reached a concentration of 16 M, whereas the antiproliferative effects were
observed at lower concentrations.
34
CA 02599884 2007-09-12
The and-proliferative effect demonstrated with A459 cells can be
similarly demonstrated using other cancer cell lines, such as MCF7 mammary
adenocarcinoma, PA-1 ovarian teratocarcinoma, HT29 colorectal
adenocarcinoma, H1299 large cell carcinoma, U-2 OS osteogenic sarcoma, U-
s 373 MG glioblastoma, Hep-3B hepatocellular carcinoma, BT-549 mammary
carcinoma, T-24 bladder cancer, C-33A cervical carcinoma, HT-3 metastatic
cervical carcinoma, SiHa squamous cervical carcinoma, CaSki epidermoid
cervical carcinoma, NCI-H292 mucoepidermoid lung carcinoma, NCI-2030,
non small cell lung carcinoma, HeLa, epithelial cervical adenocarcinoma, KB
io epithelial mouth carcinoma, HT1080 epithelial fibrosarcoma, Saos-2
epithelial
osteogenic sarcoma, PC3 epithelial prostate adenocarcinoma, SW480
colorectal carcinoma, CCL-228, and MS-751 epidermoid cervical carcinoma
cell lines. The specificity can be tested by using cells such as NHLF lung
fibroblasts, NHDF dermal fibroblasts, HMEC mammary epithelial cells, PrEC
15 prostate epithelial cells, HRE renal epithelial cells, NHBE bronchial
epithelial
cells, CoSmC Colon smooth muscle cells, CoEC colon endothelial cells,
NHEK epidermal keratinocytes, and bone marrow cells as control cells.
Other Embodiments
20 Various modifications and variations of the described method and system of
the
invention will be apparent to those skilled in the art without departing from
the scope of
the invention. Although thc invention has been described in connection with
specific
preferred embodiments, it should be understood that the invention as claimed
should
not be unduly limited to such specific embodiments. Indeed, various
modifications of
25 the described modes for carrying out the invention that are obvious to
those skilled in
molecular biology or related fields are intended to be within the scope of the
invention.
