Note: Descriptions are shown in the official language in which they were submitted.
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USE OF SUBS1TTUTED 11-PHENYITDIBENZAZEPINE COMPOUNDS FOR TIC TREA1~IENT OR
PREVENTION OF
SICKLE CELL DISEASE, INFLAMMATORY DISEASES CHARACTERIZED BY ABNORMAL CELL
PROLIFERATION,
DIARRHEA AND SCOUR
Field of the Invention
The present invention relates to aromatic organic compounds which are
specific,
potent and safe inhibitors of the Ca'-+-activated potassium channel (Gardos
channel) of
erythrocytes, of mammalian cell proliferation, and/or of secretagogue-
stimulated
to transepithelial electrogenic chloride secretion in intestinal cells. The
compounds are generally
substituted 11-phenyl-dibenzazepine compounds. The compounds can be used to
reduce
sickle erythrocyte dehydration and/or delay the occurrence of erythrocyte
sickling or
deformation in situ as a therapeutic approach towards the treatment or
prevention of sickle cell
disease. The compounds can also be used to inhibit mammalian cell
proliferation in situ as a
15 therapeutic approach towards the treatment or prevention of diseases
characterized by
abnormal cell proliferation. Furthermore, the compounds can be used to inhibit
chloride
secretion as a therapeutic approach towards the treatment of diarrhea and
scours.
~~ck~round of the Invention
2o Sickle cell disease has been recognized within West Africa for several
centuries.
Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first
genetic disease
to be understood at the molecular level. It is recognized today as the
morphological and
clinical result of a glycine to valine substitution at the No. 6 position of
the beta globin chain
(Ingrain, 1956, Nature 178:792-794). The origin of the amino acid change and
of the disease
25 state is the consequence of a single nucleotide substitution (Marotta et
al., 1977, J.J. Biol.
Chem. 252:5040-5053).
'The major source of morbidity and mortality of patients suffering from sickle
cell
disease is vascular occlusion caused by the sickled cells, which causes
repeated episodes of
pain in both acute and chronic form and also causes ongoing organ damage with
the passage
30 of time. It has long been recognized and accepted that the deformation and
distortion of sickle
cell erythrocytes upon complete deoxygenation is caused by polymerization and
intracellular
gelation of sickle hemoglobin, hemoglobin S (Hb S). The phenomenon is well
reviewed and
discussed by Eaton and Hofrichter, 1987, hod 70:1245. The intracellular
gelatin and
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polymerization of Hb S can occur at any time during erythrocyte's journey
through the
vasculature. Thus, erythrocytes in patients with sickle cell disease
containing no polymerized
hemoglobin S may pass through the microcirculation and return to the lungs
without sickling,
may sickle in the veins or may sickle in the capillaries.
The probability of each of these events is determined by the delay time for
intracellular gelation relative to the appropriate capillary transit time
(Eaton et a ., 1976,
Bl 47:621). In turn, the delay time is dependent upon the oxygenation state of
the
hemoglobin, with deoxygenation shortening the delay time. Thus, if it is
thermodynamically
impossible for intracellular gelation to take place, or if the delay time at
venous oxygen
1 o pressures is longer than about 15 seconds, cell sickling will not occur.
Alternatively, if the
delay time is between about l and 15 seconds, the red cell will likely sickle
in the veins.
However, if the delay time is less than about 1 second, red cells will sickle
within the
capillaries.
For red cells that sickle within the capillaries, a number of possible
consequent events
15 exist, ranging from no effect on transit time, to transient occlusion of
the capillary, to a more
permanent blockage that may ultimately result in ischemia or infarction of the
surrounding
cells, and in destruction of the red cell.
It has long been recognized that the cytoplasm of the normal erythrocyte
comprises
approximately 70% water. Water crosses a normal erythrocyte membrane in
milliseconds;
20 however, the loss of cell water causes an exponential increase in
cytoplasmic viscosity as the
mean cell hemoglobin concentration (MCHC) rises above about 32 g/dl. Since
cytoplasmic
viscosity is a major determinate of erythrocyte deformability and sickling,
the dehydration of
the erythrocyte has substantial rheological and pathological consequences.
Thus, the
physiological mechanisms that maintain the water content of a normal
erythrocytes and the
25 pathological conditions that cause loss of water from erythrocytes in the
blood circulation are
critically important. Not surprisingly, regulation of erythrocyte dehydration
has been
recognized as an important therapeutic approach towards the treatment of
sickle cell disease.
Since cell water will follow any osmotic change in the intracellular
concentration of ions, the
maintenance of the red cell's potassium concentration is of particular
importance (Stuart and
3o Ellory, 1988, Brit J. Haematol. ø~:1-4).
Many attempts and approaches to therapeutically treating dehydrated sickle
cells (and
thus decreasing polymerizaxion of hemoglobin S by lowering the osmolality of
plasma) have
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been tried with limited success, including the following approaches:
intravenous infusion of
distilled water (Gye et al., 1973, Am. J. Med. Sci. xøø:267-277);
administration of the
antidiuretic hormone vasopressin together with a high fluid intake and salt
restriction (Rosa et
al., 1980, M. Eng. J. Med. x:1138-1143; Charache and Walker, 1981, Blood
5$:892-896);
the use of monensin to increase the cation content of the sickle cell (Clark
et al., 1982, J in.
Invest. x:1074-1080; Fahim and Pressman, 1981, Life Sciences x:1959-1966);
intravenous
administration of cetiedil citrate (Benjamin et al., 1986, Blood ,7:1442-1447;
Berkowitz and
Orringer, 1984, ~.m. J. Hematol. x:217-223; Stuart et al., 1987, J. Clin.
Pathol.
x:1182-1186); and the use of oxpentifylline (Stuart et al., 1987, J. Clin.
Pathol.
to 40:1182-1186}.
Another approach towards therapeutically treating dehydrated sickle cells
involves the
administration of imidazole, nitroimidazole and triazole antimycotic agents
such as
Clotrimazole (U.S. Patent No. 5,273,992 to Brugnara et al.). Clotrimazole, an
imidazole-
containing antimycotic agent, has been shown to be a specific, potent
inhibitor of the Gardos
~ 5 channel of normal and sickle erythrocytes, and prevents Ca2+-dependent
dehydration of sickle
cells both in vitro and in vzvo (Brugnara et al., 1993, J. Clin. Invest. x:520-
526; De
Franceschi et al., 1994, J. Clin. Invest. x:1670-1676). When combined with a
compound
which stabilizes the oxyconformation of Hb S, Clotrimazole induces an additive
reduction in
the clogging rate of a micropore filter and may attenuate the formation of
irreversibly sickled
2o cells (Stuart et al., 1994, ,~,-Haematol. $x:820-823}. Other compounds that
contain a
heteroaryl imidazole-like moiety believed to be useful in reducing sickle
erythrocyte
dehydration via Gardos channel inhibition include miconazole, econazole,
butoconazole,
oxiconazole and sulconazole. Each of these compounds is a known antimycotic.
Other
imidazole-containing compounds have been found to be incapable of inhibiting
the Gardos
25 channel and preventing loss of potassium.
As can be seen from the above discussion, reducing sickle erythrocyte
dehydration via
blockade of the Gardos channel is a powerful therapeutic approach towards the
treatment
and/or prevention of sickle cell disease. Compounds capable of inhibiting the
Gardos channel
as a means of reducing sickle cell dehydration are highly desirable, and are
therefore an object
30 of the present invention.
Cell proliferation is a normal part of mammalian existence, necessary for life
itself.
However, cell proliferation is not always desirable, and has recently been
shown to be the root
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of many life-threatening diseases such as cancer, certain skin disorders,
inflammatory
diseases, fibrotic conditions and arteriosclerotic conditions.
Cell proliferation is critically dependent on the regulated movement of ions
across
various cellular compartments, and is associated with the synthesis of DNA.
Binding of
specific polypeptide growth factors to specific receptors in growth-arrested
cells triggers an
array of early ionic signals that are critical in the cascade of mitogenic
events eventually
leading to DNA synthesis (Rozengurt, 1986, Science x:161-164). These include
(1) a rapid
increase in cystolic Caz+, mostly due to rapid release of Ca2+ from
intracellular stores; (2)
capacitative Ca2~' influx in response to opening of ligand-bound and
hyperpolarization-
1 o sensitive Caz+ channels in the plasma membrane that contribute further to
increased
intracellular Caz+ concentration (Tsien and Tsien, 1990, Annu. Revi Cell Biol.
ø:715-760;
Peppelenbosch et al., 1991, J. Bioj~Chem. xøø:19938-19944); and (3) activation
of Ca2+-
dependent K+ channels in the plasma membrane with increased K+ conductance and
membrane hyperpolarization (Magni et al., 1991, J. Biol. Chem. 261:9321-9327).
These
~ 5 mitogen-induced early ionic changes, considered critical events in the
signal transduction
pathways, are powerful therapeutic targets for inhibition of cell
proliferation in normal and
malignant cells.
One therapeutic approach towards the treatment of diseases characterized by
unwanted
or abnormal cell proliferation via alteration of the ionic fluxes associated
with early mitogenic
2o signals involves the administration of Clotrimazole. Clotrirnazole has been
shown to inhibit
the Ca2+-activated potassium channel of erythrocytes. In addition,
Clotrimazole inhibits
voltage- and ligand-stimulated Ca2+ influx mechanisms in nucleated cells
(Villalobos et al.,
1992, FASEB J. x:2742-2747; Montero et al., 1991, B_j~QChem. J. x:73-79) and
inhibits cell
proliferation both in vitro and in vivo (Benzaquen et al., 1995, Nature M~~'c~
ine 1_:534-540).
25 Recently, Clotrimazole and other imidazole-containing antimycotic agents
capable of
inhibiting Ca2+-activated potassium channels have been shown to be useful in
the treatment of
arteriosclerosis (U.S. Patent No. 5,358,959 to Halperin et al.), as well as
other disorders
characterized by unwanted or abnormal cell proliferation.
As can be seen from the above discussion, inhibiting mammalian cell
proliferation via
3o alteration of ionic fluxes associated with early mitogenic signals is a
powerful therapeutic
approach towards the treatment andlor prevention of diseases characterized by
unwanted or
abnormal cell proliferation. Compounds capable of inhibiting mammalian cell
proliferation
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are highly desirable, and are therefore also an object of the present
invention.
Acute and chronic diarrheas represent a major medical problem in many areas of
the
world. Diarrhea is both a significant factor in malnutrition and the leading
cause of death
(5,000,000 deaths/year} in children less than five years old. Secretory
diarrheas are also a
dangerous condition in patients of acquired immunodeficiency syndrome (AIDS)
and chronic
inflammatory bowel disease {IBD}. 16 million travelers to developing countries
from
industrialized nations every year develop diarrhea, with the severity and
number of cases of
diarrhea varying depending on the country and area of travel. The major
medical
consequences of diarrheal diseases include dehydration, acidosis, death and
impaired growth.
1o Diarrhea in barn animals and pets such as cows, pigs and horses, sheep,
goats, cats and
dogs, also known as scours, is a major cause of death in these animals.
Diarrhea can result
from any major transition, such as weaning or physical movement. One form of
diarrhea is
characterized by diarrhea in response to a bacterial or viral infection and
generally occurs
within the first few hours of the animal's life.
Although the major consequences of diarrheal diseases are very similar, there
are
numerous causes of diarrhea. Secretory and exudative diarrhea are primarily
caused by
bacterial or viral infections. The most common diarrheal causing bacteria is
enterotoxogenic
E-coli (ETEC) having the K99 pilus antigen. Common viral causes of diarrhea
include
rotavirus and coronavirus. Other infectious agents include cryptosporidium,
giardia lamblia,
2o and salmonella, among others.
The treatment for diarrhea depends on the patient and the infection source.
Diarrhea
which is found in travelers to industrialized nations (travelers diarrhea)
frequently is caused
by bacterial pathogens which are acquired through ingestion of fecally
contaminated food
and/or water. Approximately 50-75% of these cases are attributed to ETEC.
Although
traveler's diarrhea is painful, it is generally not life-threatening and often
the symptoms last
only 3-5 days. The symptoms include urgent diarrhea, abdominal cramps, nausea
and fever.
The most effective course of treatment for traveler's diarrhea is the
administration of
antibiotics in conjunction with oral rehydration. It has been shown that
prophylactic
administration of antibiotics drastically reduces the number of travelers
experiencing
3o symptoms of diarrhea. However, routine administration of antibiotics is not
suggested as it
may cause resistant strains of a bacteria to develop. Other treatment methods
include
administration of bismuth subsalicylate, often taken in the form of Pepto-
Bismal,
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diphenoxylate and loperarnide.
Diarrhea in AIDS patients is a very serious condition which causes wasting and
may
be an important factor in the decline of these patients. AIDS patients often
develop diarrhea
due to enteric infections which their immune system is not capable of fighting
off, but AIDS
patients may also develop diarrhea by AIDS enteropathy. AIDS enteropathy is a
disorder
characterized by diarrhea without the involvement of secondary infections. It
is caused by the
human immunodeficiency virus (HIV) infection of the small bowel mucosal cells
and colonic
mucosal cells. The most common infective agent causing diarrhea due to enteric
infection in
AIDS patients in cryptosporidium. The methods for treating diarrhea in AIDS
patients
1 o include administration of antibiotics and administration of
immunoglobulins or an
immunoglobulin enriched fraction of bovine colostrum. Colostrum, which is the
first milk
produced by mammals after birthing is enriched with antibodies.
Acute diarrhea or scours, is a main cause of death in many newborn barn
animals such
as calves and pigs. Scours is often caused by ETEC with a K99 pilus antigen.
Infection with
the ETEC causes hypersecretion of fluid and electrolytes. Hypersecretion in
turn causes
dehydration and pH imbalance which may result in death of the newborn calf or
pig.
Newborn barn animals are also susceptible to viral infectious agents causing
scours.
Infections with rotavirus and coronavirus are common in newborn calves and
pigs. Rotavirus
infection often occurs within 12 hours of birth. Symptoms of rotaviral
infection include
2o excretion of watery feces, dehydration and weakness. Coronavirus which
causes a more
severe illness in the newborn animals, has a higher mortality rate than
rotaviral infection.
Often, however, a young animal may be infected with more than one virus or
with a
combination of viral and bacterial microorganisms at one time. This
dramatically increases
the severity of the disease.
Generally the best protection for a newborn barn animal from viral or
bacterial
infection is the consumption of colostrum. If the mother animal has been
exposed to these
infectious agents then the colostrum will contain antibodies, which are often
sufficient to
protect the newborn from contracting the diseases. Sometimes, however, this is
not sufficient
and the animals need further protection. A common method of treatment includes
3o administration of a concentrated colostrum solution or an immunoglobulin
fraction isolated
from a colostrum solution. This oral treatment may be combined with
rehydration salts.
Although these methods have improved the morbidity and mortality rate of
newborn animals
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having scours, there still exists a need for more effective treatments.
Certain imidazoles such as clotrimazole are agents which have been used both
topically and systemically as antifungals. More recently, studies have
identified other uses for
such imidazoles. U.S. patent no. 5,273,992 revealed that these imidazoles
regulate Ca++
s activated K+ channels in erythrocytes, and are thus useful in treating
sickle cell anemia, which
involves the inhibition of potassium transport. These imidazoles have also
been found to be
effective in inhibiting endothelial and/or vascular smooth muscle cell
proliferation. The
results of this finding are described in U.S. patent no. 5,358,959 and U.S.
serial no.
08/018,840, which discloses using clotrimazole for treating atherosclerotic
and angiogenic
conditions, respectively. Nonimidazole metabolites and analogs of the
foregoing compounds
also have been described as useful in treating the foregoing conditions (see
U.S. serials nos.
08/307,874 and 08/307,887).
Summary of the Invention
t s These and other objects are provided by the present invention, which in
one aspect
provides a class of organic compounds which are potent, selective and safe
inhibitors of the
CaZ+-activated potassium channel (Gardos channel) of erythrocytes, of
mammalian cell
proliferation and/or of secretagogue-stimulated transepithelial electrogenic
chloride secretion
in intestinal cells. The compounds can be used to reduce sickle erythrocyte
dehydration
2o and/or delay the occurrence of erythrocyte sickling or deformation in situ
as a therapeutic
approach towards the treatment or prevention of sickle cell disease. The
compounds can also
be used to inhibit mammalian cell proliferation in situ as a therapeutic
approach towards the
treatment or prevention of diseases characterized by abnormal cell
proliferation. Furthermore,
the compounds can also be used to inhibit chloride secretion in intestinal
cells as a therapeutic
25 approach towards the treatment of diarrhea and scours. The compounds are
generally
substituted 11-phenyl-dibenzazepine compounds. In one illustrative embodiment,
the
compounds capable of inhibiting the Gardos channel, mammalian cell
proliferation and/or
secretagogue-stimulated transepithelial electrogenic chloride secretion in
intestinal cells
according to the invention, are compounds having the structural formula (I):
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_g_
- R~s
R~
R~ i
Ra R2
Rio / R~4
R~~ ~ R~3
R~2
or pharmaceutically acceptable salts of hydrates thereof, wherein:
R, is -R', (C6-CZa) aryl or substituted (C6-C2~) aryl;
R2 is -R', -OR', -SR', halogen or trihalomethyl;
R3 is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with
R4, is (C6-
CZO) aryleno;
R, is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with
R3, is (C6-
Czo) aryleno;
each of R5, Rb, R.,, R8, R9, R,o, R", R,2, R,3 and R,4 is independently
selected from the
1 o group consisting of -R', halogen and trihalomethyl;
R,5 is -R", -C(O)R", -C(S)R", -C(O)OR", -C(S)OR", -C(O)SR", -C(S)SR",
-C(O)N(R")~, -C(S)N(R")2, -C(O)C(O)R", -C(S)C(O)R", -C{O)C(S)R", -C{S)C(S)R",
-C(O)C(O)OR", -C(S)C(O)OR", -C(O)C(S)OR", -C(O)C(O)SR", -C(S)C(S)OR",
-C(S)C(O)SR", -C(O)C(S)SR", -C(S)C(S)SR", -C(O)C(O)N(R")2, -C(S)C(O)N(R")2,
1 s -C(O)C(S)N(R")z or -C(S)C(S)N(R")z;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl and (C,-C6) alkynyl;
each R" is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (C6-C,o) aryl, substituted (C6-CZO) aryl,
(C6-CZ6) alkaryl and
2o substituted (C6-C26) alkaryl; and
the aryl and alkaryl substituents are each independently selected from the
group
consisting of -CN, -OR', -SR', -NO2, -NR'R', halogen, (C,-C6) alkyl, (C,-C6)
alkenyl, (C,-C6)
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alkynyl and trihalomethyl.
In a preferred embodiment of the invention, the chalcogens in the compounds of
formula (I) are each oxygen.
In another preferred embodiment, the compounds are those of structure (I)
wherein the
halogens are each independently -F, -Cl, -Br or -I.
In another preferred embodiment, the alkyl, alkenyl and alkynyl groups are
each
independently (C,-C3) and/or the aryl groups are phenyl and/or the aryleno
groups are
benzeno.
In another preferred embodiment, RS, Rb, R.,, R,, R,o, R" and R,3 are each
independently -R'.
In another preferred embodiment, the substituted aryl and alkaryl are mono-
substituted.
In another preferred embodiment, R,5 is -R", -C(O)R", -C(O)OR", -C(O)N(R")Z,
-C(O)C(O)R", -C(O)C(O)OR" or -C(O)C(O)N(R")z.
In another preferred embodiment of the invention, the compounds are those of
structural formula (I) wherein: R, is -R' or (C6-CZO) aryl; RZ is -R' or -OR';
R3 is -R' or -OR' or,
when taken together with R4, is (C6-Czo) aryleno; R4 is -R' or -OR' or, when
taken together
with R3, is (C6-CZO) aryleno; each of R5, Rb, R.,, Re, Rg, R,o, R", R,Z, R,3
and R,4 is
independently selected from the group consisting of -R' and halogen; R,5 is -
R", -C(O)R",
-C(O)OR", -C(O)N(R")z, -C(O)C(O)R", -C(O)C(O)OR" or -C(O)C(O)N(R")2; each R'
is
independently selected from the group consisting of -H, (C,-C6) alkyl, (C,-C6)
alkenyl and
(C,-C6) alkynyl; each R" is independently selected from the group consisting
of -H, (C,-C6)
alkyl, {C,-C6) alkenyl, (C,-C6) alkynyl, (C6-CZO) aryl, substituted (C6-Czo)
aryl, (C6-Cz6) alkaryl
and substituted (C6-C26) alkaryl; and the aryl and alkaryl substituents are
each independently
selected from the group consisting of -CN, -OR', -NOz, -NR'R', halogen, (C,-
C6) alkyl,
(C,-C6) alkenyl and (C,-C6) alkynyl.
In another preferred embodiment, the compounds are those of formula (I)
wherein: R,
is -R' or (C6-CIO) aryl; RZ is -R' or -OR'; R3 is -R' or -OR' or, when taken
together with R,4, is
(C6-C,o) aryleno; R4 is -R' or -OR' or, when taken together with R3, is (C6-
C,o) aryleno; each of
3o R5, R~ and R~ is -H; R8 is -R', -F, -Cl, -Br or -I; each of R,, R,o and R"
is -H; R,~ is -R', -F,
-Cl, -Br or -I; R,3 is -H; R,4 is -R', -F, -Cl, -Br or -I; R,5 is -R", -
C(O)R", -C(O)OR",
-C(O)N(R")2, -C(O)C(O)R", -C(O)C(O)OR" or -C(O)C(O)N(R")z; each R' is
independently
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selected from the group consisting of -H, (C,-C3) alkyl, (C,-C3) alkenyl and
(C,-C3) alkynyl;
each R" is independently selected from the group consisting of -H, (C,-C3)
alkyl, (C,-C3)
alkenyl, (C,-C3) alkynyl, (C6-C,o) aryl, substituted (C6-C,o) aryl, (C6-C,3)
alkaryl or substituted
(C6-C,3) alkaryl; and the aryl and alkaryl substituents are each independently
selected from the
group consisting of -OR',-NOz, -NR'R', -F, -Cl, -Br, -I, (C,-C3) alkyl, (C,-
C3) alkenyl and
(C,-C3) alkynyl.
In still another preferred embodiment, the compounds are those of structural
formula
(I) wherein: R, is -R' or phenyl; RZ is -R' or -OR'; R3 is -R' or -OR' or,
when taken together
with R4, is benzeno; R4 is -R' or -OR' or, when taken together with R3, is
benzeno; each of R5,
to R6 and R~ is -H; R8 is -R', -Cl or -Br; each of Rg, R,o and R" is -H; R,Z
is -R', -F or -Cl; R,3 is
-H; R,4 is -R' or -Cl; R,5 is -R", -C(O)R", -C(O)OR", -C(O)NHR", -C(O)C(O)R"
or
-C(O)C(O)OR"; each R' is independently selected from the group consisting of -
H, (C,-C3)
alkyl, (C,-C3) alkenyl and (C,-C3) alkynyl; each R" is independently selected
from the group
consisting of -H, (C,-C3) alkyl, (C,-C3) alkenyl, (C,-C3) alkynyl, (C6-C,o)
aryl, mono-
substituted {C6-C,o) aryl, (C6-C,3) alkaryl or mono-substituted (C6-C,3)
alkaryl; and the aryl
and alkaryl substituents are each independently selected from the group
consisting of -OR',
-NOZ, -NR'R', -Cl, (C,-C3) alkyl, (C,-C3) alkenyl and (C,-C3)alkynyl.
In still another aspect, the invention provides a method for reducing sickle
erythrocyte
dehydration and/or delaying the occurrence of erythrocyte sickling or
deformation in situ.
2o The method involves contacting a sickle erythrocyte in situ with an amount
of at least one
compound according to the invention, or a pharmaceutical composition thereof,
effective to
reduce sickle erythrocyte dehydration and/or delay the occurrence of
erythrocyte sickling or
deformation. In a preferred embodiment, the sickle cell dehydration is reduced
and
erythrocyte deformation is delayed in a sickle erythrocyte that is within the
microcirculation
vasculature of a subject, thereby preventing or reducing the vaso-occlusion
and consequent
adverse effects that are commonly caused by sickled cells.
In still another aspect, the invention provides a method for the treatment
and/or
prevention of sickle cell disease in a subject, such as a human. The method
involves
administering a prophylactically or therapeutically effective amount of at
least one compound
according to the invention, or a pharmaceutical composition thereof, to a
patient suffering
from sickle cell disease. The patient may be suffering from either acute
sickle crisis or
chronic sickle cell episodes.
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In one aspect of the invention a method is provided for inhibiting unwanted
cellular
proliferation associated with an inflammatory disease. The method includes the
step of
contacting a cell the proliferation of which contributes to inflammation in
situ with an amount
of a compound having the above described formula (I) effective to inhibit
proliferation of the
cell. In one embodiment the method of administration is selected from the
group consisting of
oral, parenterai, intravenous, subcutaneous, transdermal and transmucosal for
a living human.
In one embodiment the mammalian cell is a fibrotic cell or a lymphocyte.
According to another aspect of the invention a method is provided for treating
or
preventing an inflammatory disease. The method includes the step of
administering to a
1 o subject in need of such treatment a therapeutically effective amount of a
compound of the
above-described formula (I). In one embodiment the inflammatory disease is
diarrhea.
Preferably the diarrhea is caused by inflammatory bowel disease. In another
embodiment the
inflammatory disease is an autoimmune disease. In other embodiments the
inflammatory
disease is selected from the group consisting of proliferative
glomerulonephritis; lupus
erythematosus; scleroderma; temporal arteritis; thromboangiitis obliterans;
mucocutaneous
lymph node syndrome; asthma; host versus graft; inflammatory bowel disease;
multiple
sclerosis; rheumatoid arthritis; thyroiditis; Grave's disease; antigen-induced
airway
hyperactivity; pulmonary eosinophilia; Guillain-Barre syndrome; allergic
rhinitis; myasthenia
gravis; human T-lymphotrophic virus type 1-associated myelopathy; herpes
simplex
encephalitis; inflammatory myopathies; atherosclerosis; and Goodpasture's
syndrome. In
certain embodiments the administration is parenteral or per oral.
In yet another aspect, the invention provides a method for inhibiting
mammalian cell
proliferation in situ. Preferably, the mammalian cell proliferation is not
associated with a
proliferative disease selected from the group consisting of cancer, actinic
keratosis, and
Kaposi's sarcoma. The method involves contacting a mammalian cell in situ with
an amount
of at least one compound according to the invention, or a pharmaceutical
composition thereof,
effective to inhibit cell proliferation. The compound or composition may act
either
cytostatically, cytotoxically or a by a combination of both mechanisms to
inhibit proliferation.
Mammalian cells in this manner include vascular smooth muscle cells,
fibroblasts and
3o endothelial cells.
In still another aspect, the invention provides a method for treating and/or
preventing
unwanted or abnormal cell proliferation in a subject, such as a human.
Preferably, the
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unwanted or abnormal cell proliferation is not associated with a proliferative
disease selected
from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
In the method,
at least one compound according to the invention, or a pharmaceutical
composition thereof, is
administered to a subject in need of such treatment in an amount effective to
inhibit the
unwanted or abnormal mammalian cell proliferation. The compound and/or
composition may
be applied locally to the proliferating cells, or may be administered to the
subject
systemically. Preferably, the compound and/or composition is administered to a
subject that
has a disorder characterized by unwanted or abnormal cell proliferation, and
preferably the
unwanted or abnormal cell proliferation is not associated with a proliferative
disease selected
1 o from the group consisting of cancer, actinic keratosis, and Kaposi's
sarcoma. Such disorders
include, but are not limited to, non-cancerous angiogenic conditions or
arteriosclerosis.
In yet another aspect, the invention provides a method for the treatment
andlor
prevention of diseases that are characterized by unwanted and/or abnormal
mammalian cell
proliferation. Preferably, the unwanted or abnormal cell proliferation is not
associated with a
proliferative disease selected from the group consisting of cancer, actinic
keratosis, and
Kaposi's sarcoma. The method involves administering a prophylactically or
therapeutically
effective amount of at least one compound according to the invention, or a
pharmaceutical
composition thereof, to a subject in need of such treatment. Diseases that are
characterized by
abnormal mammalian cell proliferation which can be treated or prevented by way
of the
2o methods of the invention include, but are not limited to, blood vessel
proliferative disorders,
fibrotic disorders and arteriosclerotic conditions.
According to another aspect of the invention, a method for treating diarrhea
of diverse
etiology is provided. The method involves administering to a subject who is in
need of such
treatment, an aromatic compound of the invention in an amount effective to
inhibit the
diarrhea. Preferably the compound is administered orally in conjunction with
oral rehydration
fluids. The aromatic compounds useful in the invention are substituted 11-
phenyl-
dibenzazepine, or analogues thereof. In one illustrative embodiment, the
aromatic compounds
according to the invention are compounds having the above-described formula
(I). According
to one embodiment of the invention the subject in need of such treatment is a
subject who has
symptoms of diarrhea or scours. In another embodiment of the invention, the
subject in need
of such treatment is a subject at risk of developing diarrhea or scours.
In general diarrhea is a secretory disorder, which is caused by at least one
of several
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mechanisms. In one embodiment the diarrhea is an exudative form of diarrhea;
In one
embodiment the diarrhea is a nonexudative form of diarrhea; In another
embodiment the
diarrhea is a decreased absorption form of diarrhea; In another embodiment the
diarrhea is a
non-decreased absorption form of diarrhea; In yet another embodiment the
diarrhea is a
secretory form of diarrhea. In yet another embodiment the diarrhea is a
nonsecretory form of
diarrhea. In still another embodiment the diarrhea is a noninflammatory form
of diarrhea.
In another aspect, the present invention provides pharmaceutical compositions
comprising one or more compounds according to the invention in admixture with
a
pharmaceutically acceptable carrier, excipient or diluent. Such a preparation
can be
administered in the methods of the invention.
According to another aspect of the invention, pharmaceutical preparations are
provided, comprising one or more of the aromatic compounds of the invention in
admixture
with a pharmaceutically acceptable carrier, excipient or diluent, wherein the
aromatic
compounds) of the invention is (are) in an amount effective for treating: (i)
unwanted or
abnormal cell proliferation, preferably not a proliferative disease selected
from the group
consisting of cancer, actinic keratosis, and Kaposi's sarcoma; (ii) an
inflammatory disease;
(iii) sickle cell disease; and (iv) diarrhea or scours. In one embodiment, the
aromatic
compounds useful according to the invention have the general formula (I)
provided above. In
certain other embodiments, the pharmaceutical preparations include the
aromatic compounds
of the invention together with a non-formula (I) agent selected from the group
consisting of an
anti-proliferative agent; (ii) an anti-inflammatory agent; (iii) anti-sickle
cell agent; and (iv) an
anti-diarrhea or anti-scours agent.
According to another aspect, the use of aromatic compounds of the invention in
the
manufacture of medicaments is provided. The medicaments are useful for
treating: (i)
unwanted or abnormal cell proliferation, preferably not a proliferative
disease that includes
cancer, actinic keratosis, and Kaposi's sarcoma; (ii) an inflammatory disease;
(iii) sickle cell
disease; and (iv) diarrhea or scours.
According to another aspect of the invention, pharmaceutical preparations are
provided. These pharmaceutical preparations include the aromatic compounds of
the
3o invention together with an anti-diarrheal agent. In one embodiment, the
aromatic compounds
useful according to the invention have the general formula (I) provided above.
In other
embodiments the aromatic compounds useful according to the invention are the
preferred
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compounds described above. Preferably the pharmaceutical.composition of the
invention may
be administered orally.
The invention also provides the aromatic compounds of the invention in the
manufacture of a medicament for the treatment of diarrhea or scours. In one
embodiment, the
aromatic compounds useful according to the invention have the general formula
(I) provided
above. In other embodiments the aromatic compounds useful according to the
invention are
the preferred compounds described above.
According to another aspect of the invention, veterinary preparations are
provided.
These veterinary preparations include the aromatic compounds useful according
to the
to invention together with an anti-scours preparation. In one embodiment, the
aromatic
compounds useful according to the invention have the general formula (I)
provided above. In
other embodiments the aromatic compounds useful according to the invention are
the
preferred compounds described above.
Each of the limitations of the invention can encompass various embodiments of
the
invention. It is, therefore, anticipated that each of the limitations of the
invention involving
any one element or combinations of elements can be included in each aspect of
the invention.
brief Description of the Drawings
2o FIG. 1 is a general reaction scheme for synthesizing certain compounds
according to
the invention;
FIG. 2 is a general reaction scheme for synthesizing certain compounds
according to
the invention;
FIG. 3 is a bar graph depicting the effect of clotrimazole in the inhibition
of cAMP and
Ca++ dependent Cl- secretion in T84 cells; and
FIG. 4 is a graph showing the effect of clotrimazole on the inhibition of base
line and
Ca++ - stimulated 86 Rb efflux from T84 monolayers.
Detailed Description of the Invention
3o As discussed in the Background section, blockade of sickle dehydration via
inhibition
of the Gardos channel is a powerful therapeutic approach towards the treatment
and/or
prevention of sickle cell disease. In vitro studies have shown that
Clotrimazole, an imidazole-
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containing antimycotic agent, blocks Ca''-activated K+ transport and cell
dehydration in sickle
erythrocytes (Brugnara et al., 1993, J. Clin. Invest. x:520-526). Studies in a
transgenic
mouse model for sickle cell disease (SAD mouse, Trudel et al., 1991, O J.
11:3157-
3165) show that oral administration of Clotrimazole leads to inhibition of the
red cell Gardos
s channel, increased red cell K+ content, a decreased mean cell hemoglobin
concentration
(MCHC) and decreased cell density (De Franceschi et al., 1994, J. Clin.
Invest. 23:1670-
1676). Moreover, therapy with oral Clotrimazole induces inhibition of the
Gardos channel
and reduces erythrocyte dehydration in patients with sickle cell disease
(Brugnara et al., 1996,
J. Clin. Invest. X7:1227-1234). Other antimycotic agents which inhibit the
Gardos channel in
Io vitro include miconazole, econazole, butoconazole, oxiconazole and
sulconazole (U.S. Patent
No. 5,273,992 to Brugnara et al.). All of these compounds contain an imidazole-
like ring, i.e.,
a heteroaryl ring containing two or more nitrogens.
Also as discussed in the Background section, the modulation of early ionic
mitogenic
signals and inhibition of cell proliferation are powerful therapeutic
approaches towards the
~ s treatment and/or prevention of disorders characterized by abnormal cell
proliferation. It has
been shown that Clotrimazole, in addition to inhibiting the Gardos channel of
erythrocytes,
also modulates ionic mitogenic signals and inhibits cell proliferation both in
vitro and in vivo.
For example, Clotrimazole inhibits the rate of cell proliferation of normal
and cancer
cell lines in a reversible and dose-dependent manner in vitro (Benzaquen etet
al., 1995 a r
2o Medicine x:534-540). Clotrimazole also depletes the intracellular Caz+
stores and prevents the
rise in cystolic Ca2+ that normally follows mitogenic stimulation. Moreover,
in mice with
severe combined immunodeficiency disease (SCID) and inoculated with MM-RU
human
melanoma cells, daily administration of Clotrimazole resulted in a significant
reduction in the
number of lung metastases observed (Benzaquen et al., supra).
2s The discovery that 11-phenyl dibenzazepine compounds, and analogues
thereof,
inhibit the Gardos channel of erythrocytes, mammalian cell proliferation
and/or Cl- secretion
from intestinal cells, was quite surprising. Thus, in one aspect, the present
invention provides
a new class of organic compounds that are capable of inhibiting the Gardos
channel of
erythrocytes, mammalian cell proliferation, particularly mitogen-induced cell
proliferation,
3o and/or CY secretion from intestinal cells.
Significantly, the compounds of the invention do not contain an imidazole or
imidazole-like moiety. The imidazole or imidazole-like moiety is well-
recognized as the
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essential functionality underlying the antimycotic and other biological
activities of
Clotrimazole and the other above-mentioned anti-mycotic agents. Thus, the 11-
phenyl
dibenzazepine compounds of the invention provide an entirely new class of
compounds that
are capable of effecting inhibition of the Gardos channel of erythrocytes,
mammalian cell
proliferation, particularly mitogen-induced cell proliferation, and/or Cl-
secretion from
intestinal cells.
In another aspect, the invention provides a method of reducing sickle cell
dehydration
and/or delaying the occurrence of erythrocyte sickling in situ as a
therapeutic approach
towards the treatment of sickle cell disease. In its broadest sense, the
method involves only a
i 0 single step -- the administration of at least one pharmacologically active
compound of the
invention, or a composition thereof, to a sickle erythrocyte in situ in an
amount effective to
reduce dehydration and/or delay the occurrence of cell sickling or
deformation.
While not intending to be bound by any particular theory, it is believed that
administration of the active compounds described herein in appropriate amounts
to sickle
erythrocytes in situ causes nearly complete inhibition of the Gardos channel
of sickle cells,
thereby reducing the dehydration of sickle cells and/or delaying the
occurrence of cell sickling
or deformation. In a preferred embodiment, the dehydration of a sickle cell is
reduced and/or
the occurrence of sickling is delayed in a sickle cell that is within the
microcirculation
vasculature of the subject, thereby reducing or eliminating the vaso-occlusion
that is
2o commonly caused by sickled cells.
Based in part on the surmised importance of the Gardos channel as a
therapeutic target
in the treatment of sickle cell disease, the invention is also directed to
methods of treating or
preventing sickle cell disease. In the method, an effective amount of one or
more compounds
according to the invention, or a pharmaceutical composition thereof, is
administered to a
patient suffering from sickle cell disease. The methods may be used to treat
sickle cell disease
prophylactically to decrease intracellular Hb S concentration and/or
polymerization, and thus
diminish the time and duration of red cell sickling and vaso-occlusion in the
blood circulation.
The methods may also be used therapeutically in patients with acute sickle
cell crisis, and in
patients suffering chronic sickle cell episodes to control both the frequency
and duration of
3o the crises.
The compounds of the invention are also potent, specific inhibitors of
mammalian cell
proliferation. Thus, in another aspect, the invention provides methods of
inhibiting
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mammalian cell proliferation as a therapeutic approach towards the treatment
or prevention of
diseases characterized by unwanted or abnormal cell proliferation. In its
broadest sense, the
method involves only a single step -- the administration of an effective
amount of at least one
pharmacologically active compound according to the invention to a mammalian
cell in situ.
The compound may act cytostatically, cytotoxically, or by a combination of
both mechanisms
to inhibit cell proliferation. Mammalian cells treatable in this manner
include vascular
smooth muscle cells, fibroblasts, endothelial cells, various pre-cancer cells
and various cancer
cells. In a preferred embodiment, cell proliferation is inhibited in a subject
suffering from a
disorder that is characterized by unwanted or abnormal cell proliferation.
Such diseases are
1 o described more fully below.
Based in part on the surmised role of mammalian cell proliferation in certain
diseases,
the invention is also directed to methods of treating or preventing diseases
characterized by
abnormal cell proliferation. In the method, an effective amount of at least
one compound
according to the invention, or a pharmaceutical composition thereof, is
administered to a
~ 5 patient suffering from a disorder that is characterized by abnormal cell
proliferation. While
not intending to be bound by any particular theory, it is believed that
administration of an
appropriate amount of a compound according to the invention to a subject
inhibits cell
proliferation by altering the ionic fluxes associated with early mitogenic
signals. Such
alteration of ionic fluxes is thought to be due to the ability of the
compounds of the invention
20 to inhibit potassium channels of cells, particularly Ca2+-activated
potassium channels. The
method can be used prophylactically to prevent unwanted or abnormal cell
proliferation, or
may be used therapeutically to reduce or arrest proliferation of abnormally
proliferating cells.
The compound, or a pharmaceutical formulation thereof, can be applied locally
to
proliferating cells to arrest or inhibit proliferation at a desired time, or
may be administered to
25 a subject systemically to arrest or inhibit cell proliferation.
Diseases which are characterized by abnormal cell proliferation that can be
treated or
prevented by means of the present invention include blood vessel proliferative
disorders,
fibrotic disorders, arteriosclerotic disorders and various cancers.
Blood vessel proliferation disorders refer to angiogenic and vasculogenic
disorders
3o generally resulting in abnormal proliferation of blood vessels. The
formation and spreading
of blood vessels, or vasculogenesis and angiogenesis, respectively, play
important roles in a
variety of physiological processes such as embryonic development, corpus
luteum formation,
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wound healing and organ regeneration. They also play a pivotal role in cancer
development.
Other examples of blood vessel proliferative disorders include arthritis,
where new capillary
blood vessels invade the joint and destroy cartilage and ocular diseases such
as diabetic
retinopathy, where new capillaries in the retina invade the vitreous, bleed
and cause blindness
and neovascular glaucoma.
Another example of abnormal neovascularization is that associated with solid
tumors.
It is now established that unrestricted growth of tumors is dependent upon
angiogenesis and
that induction of angiogenesis by liberation of angiogenic factors can be an
important step in
carcinogenesis. For example, basic fibroblast growth factor (bFGF) is
liberated by several
1o cancer cells and plays a crucial role in cancer angiogenesis. The
demonstration that certain
animal tumors regress when angiogenesis is inhibited has provided the most
compelling
evidence for the role of angiogenesis in tumor growth. Other cancers that are
associated with
neovascularization include hemangioendotheliomas, hemangiomas and Kaposi's
sarcoma.
Proliferation of endothelial and vascular smooth muscle cells is the main
feature of
neovascularization. The invention is useful in inhibiting such proliferation,
and therefore in
inhibiting or arresting altogether the progression of the angiogenic condition
which depends in
whole or in part upon such neovascularization. The invention is particularly
useful when the
condition has an additional element of endothelial or vascular smooth muscle
cell
proliferation that is not necessarily associated with neovascularization. For
example, psoriasis
2o may additionally involve endothelial cell proliferation that is independent
of the endothelial
cell proliferation associated with neovascularization. Likewise, a solid tumor
which requires
neovascularization for continued growth may also be a tumor of endothelial or
vascular
smooth muscle cells. In this case, growth of the tumor cells themselves, as
well as the
neovascularization, is inhibited by the compounds described herein.
The invention is also useful for the treatment of fibrotic disorders such as
fibrosis and
other medical complications of fibrosis which result in whole or in part from
the proliferation
of fibroblasts. Medical conditions involving fibrosis (other than
atherosclerosis, discussed
below) include undesirable tissue adhesion resulting from surgery or injury.
Other cell proliferative disorders which can be treated by means of the
invention
3o include arteriosclerotic conditions. Arteriosclerosis is a term used to
describe a thickening
and hardening of the arterial wall. An arteriosclerotic condition as used
herein means
classical atherosclerosis, accelerated atherosclerosis, atherosclerotic
lesions and any other
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arteriosclerotic conditions characterized by undesirable endothelial and/or
vascular smooth
muscle cell proliferation, including vascular complications of diabetes.
Proliferation of vascular smooth muscle cells is a main pathological feature
in classical
atherosclerosis. It is believed that liberation of growth factors from
endothelial cells
stimulates the proliferation of subintimal smooth muscle which, in turn,
reduces the caliber
and finally obstructs the artery. The invention is useful in inhibiting such
proliferation, and
therefore in delaying the onset of, inhibiting the progression of, or even
halting the
progression of such proliferation and the associated atherosclerotic
condition.
Proliferation of vascular smooth muscle cells produces accelerated
atherosclerosis,
to which is the main reason for failure of heart transplants that are not
rejected. This
proliferation is also believed to be mediated by growth factors, and can
ultimately result in
obstruction of the coronary arteries. The invention is useful in inhibiting
such obstruction and
reducing the risk of, or even preventing, such failures.
Vascular injury can also result in endothelial and vascular smooth muscle cell
proliferation. The injury can be caused by any number of traumatic events or
interventions,
including vascular surgery and balloon angioplasty. Restenosis is the main
complication of
successful balloon angioplasty of the coronary arteries. It is believed to be
caused by the
release of growth factors as a result of mechanical injury to the endothelial
cells lining the
coronary arteries. Thus, by inhibiting unwanted endothelial and smooth muscle
cell
2o proliferation, the compounds described herein can be used to delay, or even
avoid, the onset of
restenosis.
Other atherosclerotic conditions which can be treated or prevented by means of
the
present invention include diseases of the arterial walls that involve
proliferation of endothelial
and/or vascular smooth muscle cells, such as complications of diabetes,
diabetic
glomerulosclerosis and diabetic retinopathy.
The compounds described herein are also useful in treating or preventing
various types
of cancers. Cancers which can be treated by means of the present invention
include, but are
not limited to, biliary tract cancer; brain cancer, including glioblastomas
and
medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon
cancer; endometrial
3o cancer; esophageal cancer; gastric cancer; hematological neoplasms,
including acute and
chronic lymphocytic and myelogenous leukemia, multiple myeloma, AIDS
associated
leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms,
including Bowen's
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disease and Paget's disease; liver cancer; lung cancer; lymphomas, including
Hodgkin's
disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including
squamous cell
carcinoma; ovarian cancer, including those arising from epithelial cells,
stromal cells, germ
cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;
sarcomas,
including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and
osteosarcoma;
skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and
squamous cell
cancer; testicular cancer, including germinal tumors (seminoma, non-seminoma
(teratomas,
choriocarcinomas)), stromal tumors and germ cell tumors; thyroid cancer,
including thyroid
adenocarcinoma and medullar carcinoma; and renal cancer including
adenocarcinoma and
1 o Wilms tumor.
T'he compounds of the invention are useful with hormone dependent and also
with
nonhormone dependent cancers. They also are useful with prostate and
nonprostate cancers
and with breast and nonbreast cancers. They further are useful with multidrug
resistant strains
of cancer.
In addition to the particular disorders enumerated above, the invention is
also useful in
treating or preventing dermatological diseases including keloids, hypertrophic
scars,
seborrheic dermatosis, papilloma virus infection (e.g., producing verruca
vulgaris, verruca
plantaris, verruca plan, condylomata, etc.), and eczema and epithelial
precancerous lesions
such as actinic keratosis. It also is useful with pathologies mediated by
growth factors such as
2o uterine leiomyomas.
In addition to the particular disorders enumerated above, the invention is
particularly
useful in treating or preventing inflammatory diseases associated with
cellular proliferation.
An "inflammatory disease associated with cellular proliferation" as used
herein is a disease in
which lymphoproliferation contributes to tissue or organ damage leading to
disease. For
instance, excessive T cell proliferation at the site of a tissue or organ will
cause damage to the
tissue or organ. Inflammatory disease are well known in the art and have been
described
extensively in medical textbooks (See, e.g., Harrison's Principles of
Experimental Medicine,
13th Edition, McGraw-Hill, Inc., N.Y.).
Inflammatory diseases associated with cellular proliferation include but are
not limited
3o to proliferative glomerulonephritis; lupus erythematosus; scleroderma;
temporal arteritis;
thromboangiitis obliterans; mucocutaneous lymph node syndrome; asthma; host
versus graft;
inflammatory bowel disease; multiple sclerosis; rheumatoid arthritis;
thyroiditis; Grave's
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disease; antigen-induced airway hyperactivity; pulmonary eosinophilia;
Guillain-Barre
syndrome; allergic rhinitis; myasthenia gravis; human T-lymphotrophic virus
type 1-
associated myelopathy; herpes simplex encephalitis; inflammatory myopathies;
atherosclerosis; and Goodpasture's syndrome. Some examples of inflammatory
diseases
associated with cellular proliferation as well as animal models for testing
and developing the
compounds are set forth in Table 1 below.
Table 1
Disease ProliferatingReference Animal ModelReference
Cells
Asthma T cells Hogg 1997 Airway inflammationHenderson
APMIS et al.
100:105(IO):735-45and 1997 J Clin
Invest
hyperresponsiveness100(12):3083-3092.
in Ovalbumin-
sensitized
mice or
guinea pigs.
GlomerulonephritisMesangial Nitta et al. NZBMZW crossedClynes et
1998 al. 1998
(glomerular) Eur J Pharmacolmice developScience 279(5353):
cells
344:107-120 glomerular 1052-54.
disease
and lupus-like
syndrome.
Host versus T cells Schorlemmer Renal allograftLazarivuts
Graft et al. et al. 1996
B cells 1997 Int J rejection Nature 380(6576)
Tissue in mice.
React 19:157-61. 7I7-720.
Sedgwick et
al. 1998
J Immunol
160:5320-30.
InflammatoryEpithelial Bajaj-ElliottTrinitrobenzeneBoughton-Smith
Bowel cells et al. et
Disease 1997 Am J. sulphonic al. 1988 Br
Pathol. acid J
151:1469-76. induced bowelPharmacol
94:65-72.
inflammation
in rats.
Systemic Glomerular Kodera et NZB/NZW crossedPeng et al.
Lupus cells al. 1997 1996
ErythematosisLymphocytes Am J Nephol mice developMol Biol Rep
17:466- 23(3-
70. glomerular 4):247-51.
disease
Akashi et and lupus-like
al. 1998
Immunology syndrome.
93:238-
48
Multiple T cells ConstantinesecuExperimentalDrescher et
Sclerosis et allergic al. 1998
al. 1998 lmmunolencephalomyelitis.J Clin Invest
Res 17(1-2):217-27. 101(8):1765-74.
Rheumatoid T cells Ceponis et Rat adjuvantAnderson et
Arthritis al. 1998 arthritis al. 1996
Synovial cellsBr J Rheumatolassay J Clin Invest
37(2):170-8 97( 11 ):2672-9.
Thyroiditis T cells and Rose et al. HLA transgenicTaneja et
1997 mice al. 1998
Epithelial Crit Rev Immunolimmunized J Clin Investig
cells with
17:511-7. thyroglobulin.101(5):921-6.
Schumm-Draeger
et
al. 1996 Verh
Dtsch
Ges Pathol
80:297-
301.
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Grave's DiseaseThyroid cellsDiPaola et Thiouracil-fedViglietto
al. 1997 rats. et al. 1997
J Clin Endocrinol Oncogene 15:2687-
Metab 82:670-3. g8.
Disease Proliferating Reference Model
Celts
Antigen-inducedT cells Wolyniec et al.
airway 1998
hyperactivity Am J Respir Cell
Mol
Biol 18:777-85
Pulmonary eosinophiliaT cells Wolyniec et al.
1998
Am J Respir Cell
MoI
Biol 18:777-85
Guillain-Bane T cells Hartung et al. Experimental
1991
Syndrome Ann Neurol. 30:48-53autoimmune neuritis
(inflammatory
(immunization
with PNS
demyelinating myelin and Freunds
disease)
complete adjuvant)
Giant cell arteritisT cells Brack et al.
(a 1997
form of systemic Mol Med 3:530-43
vasculitis)
Inflammation
of large arteries
Allergic RhinitisT cells Baraniuk et al.
1997
J Allergy Clin
Immunol
99:S763-72
Myasthenia gravisT cells Hartung et al.
1991
Ann Neurol 30:48-53
Human T-lymphotropicT cells Nakamura et al.
1996
virus type 1 Intern Mede 35:195-99
- associated
myelonathy
Herpes simplex T cells Hartung et al.
1991
encephalitis Ann Neurol 30:48-53
Inflammatory T cells Hartung et al.
1991
myopathies (ie. Ann Neurol 30:48-53.
Polymyositis, Lindberg et al.
1995
detmatomyocitis) Scand J Immunol
41:421-26
ArtherosclerosisT cells Rosenfeld et
al. 1996
Diabetes Res
Clin Pract
30 suppl.: 1-11
Goodpasture's Macrophages Lan et al. 1995
syndrome
Am J Pathol 147:1214-
20
The compounds and methods of the invention provide myriad advantages over
agents
3o and methods commonly used to treat cell proliferative disorders. For
example, many of the
compounds of the invention are more potent than Clotrimazole in in vitro
tests, and therefore
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may provide consequential therapeutic advantages in clinical settings.
Most significantly, the compounds of the invention have reduced toxicity as
compared
with these other agents. For Clotrimazole, it is well-known that the imidazole
moiety is
responsible for inhibiting a wide range of cytochrome P-450 isozyme catalyzed
reactions,
which constitutes their main toxicological effects (Pappas and Franklin, 1993,
~g 'xicoloev
$Q:27-35; Matsuura et al., 1991, Biochemical Pharmacolo ~ 41:1949-1956).
Analogues and
metabolites of Clotrimazole do not induce cytochrome P-450 (Matsuura et al.,
1991,
Biochemical Pha_rma, col~~y 41:1949-1956), and therefore do not share
Clotrimazole's toxicity.
The invention in another aspect also involves methods and products for
reducing the
to symptoms of diarrhea or preventing diarrhea in a subject at risk for
developing diarrhea, using
the compounds of the invention. The aromatic compounds useful according to the
invention
may be provided in a pharmaceutical preparation or a veterinary preparation.
The aromatic
compounds of the invention are also useful in a method for treating diarrhea
and scours as
well as a method for preventing diarrhea and scours.
~ 5 Diarrhea, as used herein, indicates a medical syndrome which is
characterized by the
symptoms of diarrhea or scours. In general, diarrhea is a disorder resulting
in a secretory
imbalance. For purposes of this patent application diarrhea is divided into
three categories
based on the underlying mechanism: exudative, decreased absorption, and
secretory and the
term diarrhea as used herein encompasses each of these categories. Exudative
diarrheas result
2o from inflammatory processes leading to impaired colonic absorption, and
outpouring of cells
and colloid caused by such disorders as ulcerative colitis, shigellosis, and
amebiasis.
Disorders of decreased absorption include osmotic, anatomic derangement, and
motility
disorders. Osmotic diarrhea can occur as a result of digestive abnormalities
such as lactose
intolerance. Anatomic derangement results in a decreased absorption surface
caused by such
25 procedures as subtotal colectomy and gastrocolic fistula. Motility
disorders result from
decreased contact time resulting from such diseases as hyperthyroidism and
irritable bowel
syndrome. Secretory diarrhea is characterized by the hypersecretion of fluid
and electrolytes
from the cells of the intestinal wall. In classical form, the hypersecretion
is due to changes
which are independent of the permeability, absorptive capacity and exogenously
generated
30 osmotic gradients within the intestine. As discussed above, however, all
forms of diarrhea
may actually manifest a secretory component.
The methods and products of the invention are particularly useful in treating
diarrhea
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PCT/US98/24967
which is secretory. However, the methods and products of the invention may
also be used in
combination with other treatment methods which are known in the art to treat
diarrhea caused
by decreased absorption or inflammation. The compounds of the invention are
involved in
regulating CY secretion and can function alone or when used in combination
with other
treatment methods to decrease net fluid secretion even when this is due
primarily to
abnormalities in absorption or inflammation.
The methods and products of the invention are useful in preventing diarrhea
and
scours in subjects at risk of developing these disorders. Subjects at risk of
developing
diarrhea and scours are those subjects which have a high likelihood of
exposure to the
1o bacterial and viral microorganisms which cause these symptoms. For example,
approximately
1/3 of travelers to developing countries will develop diarrhea; infection with
rotavirus is one
of the leading causes of death in infants in developing countries; subjects
with HIV have a
greater than 50% chance of developing diarrhea, and many newborn calves and
pigs develop
scours. Subjects with inflammatory bowel disease develop recurrent diarrhea.
The methods and products of the invention are also useful in treating subjects
who
already exhibit the symptoms of diarrhea and scours. Once a subject has been
exposed to a
microorganism causing the symptoms, the subject may be treated with the
methods and
products of the present invention in order to reduce the symptoms. The
symptoms of diarrhea
include bowel irregularity, fecal fluid rich in sodium or potassium, fluid
feces, dehydration,
fever, loss of body weight, headache, anorexia, vomiting, malaise and myalgia.
The
symptoms of scours include a loss of body weight or failure to grow,
dehydration, malodorous
feces, fluid feces, feces containing pieces of partially digested milk or
semisolid material, and
feces of a yellow-white or gray color.
The Comb
The compounds which are potent, selective and safe inhibitors of Ca2+-
activated
potassium channel (Gardos channel) of erythrocytes, particularly sickle
erythrocytes,
mammalian cell proliferation, particularly mitogen-induced cell proliferation,
and/or
secretagogue-stimulated transepithelial electrogenic chloride secretion in
intestinal cells
according to the invention, are generally substituted 11-phenyl dibenzazepine
compounds.
3o In one illustrative embodiment, the compounds capable of inhibiting the
Gardos
channel, mammalian cell proliferation and/or chloride secretion in intestinal
cells according to
the invention are compounds having the structural formula (I):
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-25-
R Rts
.s ..
Rr'"~/ ~I lI~ ~~R4
(I) Re 1 Rt I .Rs
Ra R2
Ray ~ ~R~4
R~~ ~ R~3
R~2
1 o wherein:
R, is -R', (C6-CZO) aryl or substituted (C6-CZO) aryl;
Rz is -R', -OR', -SR', halogen or trihalomethyl;
R3 is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with
R4, is (C6-
Czo) aryleno;
R4 is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with
R3, is (C6-
CZa) aryleno;
each of R5, Rs, R~, R8, Rg, R,o, R", R,2, R,3 and R,4 is independently
selected from the
group consisting of -R', halogen and trih~~~methvl;
R,5 is -R", -C(O)R", -C(S)R", -C(O)OR", -C(S)OR", -C(O)SR", -C(S)SR",
-C(O)N(R")2, -C(S)N(R")2, -C(O)C(O)R", -C(S)C(O)R", -C(O)C(S)R", -C(S)C(S)R",
-C(O)C(O)OR", -C(S)C(O)OR", -C(O)C(S)OR", -C(O)C(O)SR", -C(S)C(S)OR",
-C(S)C(O)SR", -C(O)C(S)SR", -C(S)C(S)SR", -C(O)C(O)N(R")z, -C(S)C(O)N(R")2,
-C(O)C(S)N(R")Z or -C(S)C(S)N(R")2;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl and (C,-C6) alkynyl;
each R" is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (C6-CZO) aryl, (C6-CZO) substituted aryl,
(C6-C26) alkaryl and
substituted (C6-CZ6) alkaryl; and
the aryl and alkaryl substituents are each independently selected from the
group
3o consisting of -CN, -OR', -SR', -NOZ, -NR'R', halogen, (C,-C6) alkyl, (C,-
C6) alkenyl, (C,-C6)
alkynyl and trihalomethyl.
In a preferred embodiment of the invention, the compounds are those of
structure (I)
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wherein the chalcogens are each oxygen.
In another preferred embodiment, the compounds are those of structure (I)
wherein the
halogens are each independently -F, -Cl, -Br or -I.
In another preferred embodiment, the alkyl, alkenyl and alkynyl groups are
each
independently (C,-C3) and/or the aryl groups are phenyl and/or the aryleno
groups are
benzeno.
In another preferred embodiment, R5, R6, R,, R9, R,o, R" and R,3 are each
independently -R'.
In another preferred embodiment, the substituted aryl and alkaryl are mono-
1 o substituted.
In another preferred embodiment, R,5 is -R", -C(O)R", -C(O)OR", -C(O)N(R")z,
-C(O)C(O)R", -C(O)C(O)OR" or -C(O)C(O)N(R")z.
In another preferred embodiment of the invention, the compounds are those of
structural formula (I) wherein:
t 5 R, is -R' or (C6-Czo) aryl;
Rz is -R' or -OR';
R3 is -R' or -OR' or, when taken together with R4, is (C6-Czo) aryleno;
R4 is -R' or -OR' or, when taken together with R3, is (C6-Czo) aryleno;
each of R5, R6, R~, R8, Ra, R;~, p ", n~z, R,3 and R,4 is independently
selected from the
2o group consisting of -R' and halogen;
R,5 is -R", -C(O)R", -C(O)OR", -C(O)N(R")z, -C(O)C(O)R", -C(O)C(O)OR" or
-C(O)C(O)N(R")z;
each R' is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl and (C,-C6) alkynyl;
25 each R" is independently selected from the group consisting of -H, (C,-C6)
alkyl,
(C,-C6) alkenyl, (C,-C6) alkynyl, (C6-Czo) aryl, substituted (Cb-Czo) aryl,
(C6-Czb) alkaryl and
substituted (C6-Czb) alkaryl; and
the aryl and alkaryl substituents are each independently selected from the
group
consisting of -CN, -OR', -NOz, -NR'R', halogen, (C,-C6) alkyl, (C,-C6) alkenyl
and (C,-C6)
3o alkynyl.
In another preferred embodiment, the compounds are those of formula (I)
wherein:
R, is -R' or (C6-C,o) aryl;
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RZ is -R' or -OR';
Rj is -R' or -OR' or, when taken together with R,4, is (C6-C,p) aryleno;
R4 is -R' or -OR' or, when taken together with R3, is (C6-C,o) aryleno;
each of R5, R6 and R~ is -H;
R8 is -R', -F, -Cl, -Br or -I;
each of Rg, R,o and R" is -H;
R,~ is -R', -F, -Cl, -Br or -I;
R,3 is -H;
R,4 is -R', -F, -Cl, -Br or -I;
l0 R,5 is -R", -C(O)R", -C(O)OR", -C(O)N(R")z, -C(O)C(O)R", -C(O)C(O)OR" or
-C(O)C(O)N(R")2;
each R' is independently selected from the group consisting of -H, (C,-C3)
alkyl,
(C,-C3) alkenyl and (C,-C3) alkynyl;
each R" is independently selected from the group consisting of -H, (C,-C3)
alkyl,
(C,-C3) alkenyl, (C,-C3) alkynyl, (C6-C,o) aryl, substituted (C6-C,o) aryl,
(C6-C,3) alkaryl or
substituted (C6-C,3) alkaryl; and
the aryl and alkaryl substituents are each independently selected from the
group
consisting of -OR',-NOZ, -NR'R', -F, -Cl, -Br, -I, (C,-C3) alkyl, (C,-C3)
alkenyl and (C,-C3)
alkynyl.
2o In still another preferred embodiment, the compounds are those of
structural formula
(I) wherein:
R, is -R' or phenyl;
RZ is -R' or -OR';
R3 is -R' or -OR' or, when taken together with Rq, is benzeno;
R4 is -R' or -OR' or, when taken together with R3, is benzeno;
each of R5, R6 and R~ is -H;
R8 is -R', -Cl or -Br;
each of R9, R,o and R" is -H;
R,2 is -R', -F or -Cl;
3o R,3 is -H;
R,4 is -R' or -Cl;
R,5 is -R", -C(O)R", -C(O)OR", -C(O)NHR", -C(O)C(O)R" or -C(O)C(O)OR";
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each R' is independently selected from the group consisting of -H, (C,-C3)
alkyl,
(C,-C3) alkenyl and (C,-C3) alkynyl;
each R" is independently selected from the group consisting of -H, (C,-C3)
alkyl,
(C,-C3) alkenyl, (C,-C3) alkynyl, (C6-C,o) aryl, mono-substituted (C6-C,o)
aryl, (C6-C,3) alkaryl
or mono-substituted (C6-C,3) alkaryl; and
the aryl and alkaryl substituents are each independently selected from the
group
consisting of -OR', -NOz, -NR'R', -Cl, (C,-C3) alkyl, (C,-C3) alkenyl and (C,-
C,)alkynyl.
As used herein, the term "alkyl" refers to a saturated branched, straight
chain or cyclic
hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl,
isopropyl,
to cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, pentyl, isopentyl,
cyclopentyl, hexyl,
cyclohexyl and the like.
As used herein, the term "heterocycloalkyl" refers to a saturated cyclic
hydrocarbon
radical wherein one or more of the carbon atoms is replaced with another atom
such as Si, Ge,
N, O, S or P. Typical heterocycloalkyl groups include, but are not limited to,
morpholino,
15 thiolino, piperidyl, pyrrolidinyl, piperazyl, pyrazolidyl, imidazolidinyl,
and the like.
As used herein, the term "alkenyl" refers to an unsaturated branched, straight
chain or
cyclic hydrocarbon radical having at least one carbon-carbon double bond. The
radical may
be in either the cis or traps conformation about the double bond(s). Typical
alkenyl groups
include ethenyl, propenyl, isopropenyl, cyclopropenyl, butenyl, isobutenyl,
cyclobutenyl, tert-
2o butenyl, pentenyl, hexenyl and the like.
As used herein, the term "alkynyl" refers to an unsaturated branched, straight
chain or
cyclic hydrocarbon radical having at least one carbon-carbon triple bond.
Typical alkynyl
groups include ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and
the like.
As used herein, the term "alkoxy:" refers to an -OR radical, where R is alkyl,
alkenyl
25 or alkynyl, as defined above.
As used herein, the term "aryl" refers to an unsaturated cyclic hydrocarbon
radical
having a conjugated ~t electron system. Typical aryl groups include, but are
not limited to,
penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, indacenyl, and the
like.
As used herein, the term "heteroaryl" refers to an aryl group wherein one or
more of
3o the ring carbon atoms is replaced with another atom such as N, O or S.
Typical heteroaryl
groups include, but are not limited to,furanyl, imidazole, pyridinyl,
thiophenyl, indolyl,
imidazolyl, quinolyl, thienyl, indolyl, pyrrolyl, pyranyl, pyridyl, pyrimidyl,
pyrazyl,
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pyridazyl, and the like.
PCT/US98/Z4967
As used herein, the term "heteroarylium" refers to a heteroaryl group wherein
one or
more hydrogens has been added to any position of the neutral parent ring.
Typical
heteroarylium groups include, but are not limited to, pyridinium, pyrazinium,
pyrimidinium,
s pyridaziniurn, 1,3,5-triazinium, and the like.
As used herein, the term "in situ" refers to and includes the terms "in vivo,"
"ex vivo,"
and "in vitro" as these terms are commonly recognized and understood by
persons ordinarily
skilled in the art. Moreover, the phrase "in situ" is employed herein in its
broadest
connotative and denotative contexts to identify an entity, cell or tissue as
found or in place,
to without regard to its source or origin, its condition or status or its
duration or longevity at that
location or position.
In still another preferred embodiment, the compounds of the invention are as
follows:
(1) (2)
NH \ H~CN / \
l ,- ~ 1 I
N
/ C( .~. .- / 1 ~ \
CI
i
15 (3)
(4) (5)
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PCT/US98/Z4967
~OCH3
p H$C\
N
1 / \
U
CI
(6) (7)
~CH~ O CH=CHs HO
O O ~p O ~O
.~~,/N
I ~ N N
1 I ~ ~ ~ / \
CI
w ~ i i
I
(9) (10)
(m)
Ci
NH
1 / \ OCHs
O
w U O N
/ / \ ~ ~N j \
U
1
F
(12) (13)
(14)
NH / \ / ~ OCHs
1 ~ _\ ~ 0 0
N N
cl ~ I ''~ ~ 1 J ~ l J
w ~ U
(15) (16)
(I7)
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-31 -
PCT/US98I24967
NO=
NH
\ O~
\ CHa O
i
C7~ cl ~'' N
CI
\
(18) (19)
(20)
CHI
CHI
/ \ / \
O
N O
~ 4N ~ \
"~ \J
\J
cl
\ \
(21) (22)
(23)
/ \ I '-.
/ \ / \ /
N N O
-' ~ ~ ~ ~ ~ ~ \ ~ ~N ~ \
cl ci~ o \J \
\CHs ~ / CI
\ \ ~ \
(24) (25)
(26)
CHI
NO=
/ \ / \ C
0
N O
\ i ~N ~ \
1
I CI
\~
(27) (28)
(29)
CA 02310773 2000-OS-19
wo ~n66zs
rc~rnrs9sna~~
-32-
N02
N
0
~cH,
a ~ ~%'
ci
~CH3
(30) (31)
(32)
NH-CHI NHi
NH / \ NO2
O o O
N
/ 'N ~ ~ /
_ \J
ci ~ ci
i
I \
(33) (34)
(35)
The compounds will be referred to herein by way of compound numbers as
presented
above.
In yet another preferred embodiment, the compounds are those of structural
formula
(I), with the proviso that when R, and R,5 are each -R', at least one of R,,
R2, R3, R4, R5, ~,
R.,, R,, R,o, R", R~~, R,3 ar R,4 is other than -R', R8 is other than -R' or
halogen and at least
three of R2, R3, R4 and RS are other than -OR'.
In yet another preferred embodiment, the compounds are those of structural
formula
(I), with the proviso that when R, and R, 5 are each -H, at least one of R,,
R2, R3, R4, R5, ~a R.,,
R9, R,o, R", R,2, R,3 or R,4 is other than -H, Rg is other than -H or -CI and
at least three of R2,
R3, R4 and Rs are other than -OCH3.
In a final preferred embodiment, the compounds of the invention are not 11-
phenyl-
5,6-dihydro-I IH dibenz[b,e]azepine, 11-phenyl-9-halo-5,6-dihydro-11H
dibenz[b,e]azepine,
11-phenyl-9-chloro-5,6-dihydro-11H dibenz[b,e]azepine, 11-phenyl-5,6-dihydro-
1,2,3-
trialkoxy-11H dibenz(b,e]azepine, I 1-phenyl-5,6-dihydro-1,2,3-trimethoxy-I 1H
dibenz[b,e]azepine, 11-phenyl-5,6-dihydro-2,3,4-trialkoxy-I 1H
dibenz[b,e]azepine and/or 1 I-
phenyl-5,6-dihydro-2,3,4-trimethoxy-11H dibenz[b,e]azepine.
2o The chemical formulae referred to herein may exhibit the phenomena of
tautomerism
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or conformational isomerism. As the formulae drawings within this
specification can
represent only one of the possible tautomeric or conformational isomeric
forms, it should be
understood that the invention encompasses any tautomeric or conformational
isomeric forms
which exhibit biological or pharmacological activity as described herein.
The compounds of the invention may be in the form of free acids, free bases or
pharmaceutically effective salts thereof. Such salts can be readily prepared
by treating a
compound with an appropriate acid. Such acids include, by way of example and
not
limitation, inorganic acids such as hydrohalic acids (hydrochloric,
hydrobromic, etc.), sulfuric
acid, nitric acid, phosphoric acid, etc.; and organic acids such as acetic
acid, propanoic acid,
2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid,
propandioic acid,
butandioic acid, etc. Conversely, the salt can be converted into the free base
form by
treatment with alkali.
In addition to the above-described compounds and their pharmaceutically
acceptable
salts, the invention may employ, where applicable, solvated as well as
unsolvated forms of the
~5 compounds (e.g. hydrated forms).
The compounds described herein may be prepared by any processes known to
be applicable to the preparation of chemical compounds. Suitable processes are
well known
in the art. Preferred processes are illustrated by the representative
examples. Additional
methods are described in copending application Serial Number unknown, entitled
"SYNTHESIS OF 11-ARYL-5,6-DIHYDRO-11H[b,e]AZEPINES, filed Nov. 20, 1997, and
US application serial no. 08/975,594, filed November 20, 1997, entitled
"Method for the
Treatment or Prevention of Sickle Cell Disease with Substituted Phenyl-
Dibenzazepine
Compounds" which are both incorporated herein by reference in its entirety.
Necessary
starting materials may be obtained commercially or by standard procedures of
organic
chemistry. Moreover, many of the compounds are commercially available.
An individual compound's relevant activity and potency as an agent to affect
sickle
cell dehydration or deformation, mammalian cell proliferation and/or
secretagogue-stimulated
transepithelial electrogenic chloride secretion in intestinal cells may be
determined using
standard techniques. Preferentially, a compound is subject to a series of
screens to determine
3o its pharmacological activity.
In most cases, the active compounds of the invention exhibit three
pharmacological
activities: inhibition of the Gardos channel of erythrocytes, inhibition of
secretagogue-
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-34
stimulated transepithelial electrogenic chloride secretion in intestinal cells
and inhibition of
mammalian cell proliferation. However, in some cases, the compounds of the
invention may
exhibit only one of these pharmacological activities. Any compound encompassed
by formula
(I) which exhibits at least one of these pharmacological activities is
considered to be within
s the scope of the present invention.
In general, the active compounds of some embodiments of the invention are
those
which induce at least about 25% inhibition of the Gardos channel of
erythrocytes (measured at
about 10 ~M), at least about 25% inhibition of secretagogue-stimulated
transepithelial
electrogenic chloride secretion in intestinal cells (measured at about 10 ~M)
and/or about 25%
inhibition of mammalian cell proliferation (measured at about 10 pM), as
measured using in
vitro assays that are commonly known in the art (see, e.g., Brugnara et al.,
1993, J.J. Biol.
Chem. xø$(12):8760-8768; Benzaquen et al., 1995, Nature Medicine 1_:534-540).
Alternatively, or in addition, the active compounds of the invention generally
will have an
ICS° (concentration of compound that yields 50% inhibition) for
inhibition of the Gardos
1 s channel of erythrocytes of less than about 10 ~M, an ICS° for
secretagogue-stimulated
transepithelial electrogenic chloride secretion in intestinal cells of less
than about 10 pM,
andlor an ICso for inhibition of cell proliferation of less than about 10 ~.M,
as measured using
in vitro assays that are commonly known in the art (see, e.g., Brugnara et
al., 1993, T. Biol.
Chem. x($(12):8760-8768; Benzaquen et al., 1995, Nature Medicine 1_:534-540)
and the
2o Examples section below. Other assays for assessing the activity and/or
potency of an agent
with respect to the uses of the invention are described below with respect to
an effective
amount of the compounds.
Representative active compounds according to the invention include Compounds 1
through 35, as illustrated above.
25 In certain embodiments of the invention, compounds which exhibit only one
pharmacological activity, or a higher degree of one activity, may be
preferred. Thus, when
the compound is to be used in methods to treat or prevent sickle cell disease,
or in methods to
reduce sickle cell dehydration and/or delay the occurrence of erythrocyte
sickling or
deformation in situ, it is preferred that the compound exhibit at least about
75% Gardos
3o channel inhibition (measured at about 10 ~M) and/or have an ICS° of
Gardos channel
inhibition of less than about 1 ~.M, with at least about 90% inhibition and/or
an ICS° of less
than about 0.1 pM being particularly preferred.
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-35
Exemplary preferred compounds for use in methods related to Gardos channel
inhibition and sickle cell disease include Compounds 1, 2, 3, 4, 6, 9, 18, 29
and 35, with
Compounds 2, 3, 4, 6, 9, 29 and 35 being particularly preferred.
When the compound is to be used in methods to treat or prevent diarrhea and/or
scours, it is preferred that the compound exhibit at least about 75%
inhibition of CY secretion
from intestinal cells (measured at about 10 ~cM) and/or have an ICS° of
inhibition of Cl-
secretion from intestinal cells of less than about 1 pM, with at least about
90% inhibition
and/or an ICS° of less than about 0.1 pM being particularly preferred.
When the compound is to be used in methods to treat or prevent disorders
io characterized by abnormal cell proliferation or in methods to inhibit cell
proliferation in situ,
it is preferable that the compound exhibit at least about 75% inhibition of
mitogen-induced
cell proliferation (measured at about 10 uM) and/or have an ICS° of
cell proliferation of less
than about 3 pM, with at least about 90% inhibition and/or an ICSO of less
than about 1 ~.M
being particularly preferred. Even more preferred compounds meet both the %
inhibition and
t s ICS° criteria.
Exemplary preferred compounds for use in methods of inhibiting mammalian cell
proliferation or for the treatment or prevention of diseases characterized by
abnormal cell
proliferation include Compounds 2, 3, 4, 7, 8, 9,13,14, 16, 17,18, 19, 20, 21,
22, 26, 27, 28,
29, 30, 31, 32, 33 and 34, with Compounds 14, 26, 28, 29, 30 and 31 being
particularly
2o preferred.
Formulation and IZOUteS c1f At~minietratinn
The compounds described herein, or pharmaceutically acceptable addition salts
or
hydrates thereof, can be delivered to a patient using a wide variety of routes
or modes of
administration. Suitable routes of administration include, but are not limited
to, inhalation,
25 transdermal, oral, rectal, transmucosal, intestinal and parenteral
administration, including
intramuscular, subcutaneous and intravenous injections.
The compounds described herein, or pharmaceutically acceptable salts andlor
hydrates
thereof, may be administered singly, in combination with other compounds of
the invention,
and/or in cocktails combined with other therapeutic agents. Of course, the
choice of
3o therapeutic agents that can be co-administered with the compounds of the
invention will
depend, in part, on the condition being treated.
A subject as used herein, means humans, primates, horses, cows, sheep, pigs,
goats,
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cats and dogs.
For example, when administered to subjects undergoing cancer treatment, the
compounds may be administered in cocktails containing other anti-cancer agents
and/or
supplementary potentiating agents. The compounds may also be administered in
cocktails
containing agents that treat the side-effects of radiation therapy, such as
anti-emetics, radiation
protectants, etc.
Anti-cancer drugs that can be co-administered with the compounds of the
invention
include, e.g., Aminoglutethimide; Asparaginase; Bleomycin; Busulfan;
Carboplatin;
Carmustine (BCNU); Chlorambucil; Cisplatin (cis-DDP); Cyclophosphamide;
Cytarabine
1 o HCI; Dacarbazine; Dactinomycin; Daunorubicin HCI; Doxorubicin HCI;
Estramustine
phosphate sodium; Etoposide (VP-16); Floxuridine; Fluorouracil (5-FU);
Flutamide;
Hydroxyurea (hydroxycarbamide); Ifosfamide; Interferon Alfa-2a, Alfa 2b,
Lueprolide acetate
(LHRH-releasing factor analogue); Lomustine (CCNU); Mechlorethamine HCl
(nitrogen
mustard); Melphalan; Mercaptopurine; Mesna; Methotrexate (MTX); Mitomycin;
Mitotane
~ 5 (o.p'-DDD); Mitoxantrone HCI; Octreotide; Plicamycin; Procarbazine HCI;
Streptozocin;
Tamoxifen citrate; Thioguanine; Thiotepa; Vinblastine sulfate; Vincristine
sulfate; Amsacrine
(m-AMSA); Azacitidine; Hexamethylmelamine (HMM); Interleukin 2; Mitoguazone
(methyl-
GAG; methyl glyoxal bis-guanylhydrazone; MGBG); Pentostatin; Semustine (methyl
CCNU); Teniposide (VM-26); paclitaxel and other taxanes; and Vindesine
sulfate.
20 Supplementary potentiating agents that can be co-administered with the
compounds of
the invention include, e.g., Tricyclic anti-depressant drugs (e.g.,
imipramine, desipramine,
amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline,
protriptyline, amoxapine
and maprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline,
trazodone and
citalopram); Ca++ antagonists (e.g., verapamil, nifedipine, nitrendipine and
caroverine);
25 Amphotericin (e.g., Tween 80 and perhexiline maleate); Triparanol analogues
(e.g.,
tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs
(e.g., reserpine);
Thiol depleters (e.g., buthionine and sulfoximine); and calcium leucovorin.
The active compounds) may be administered per se or in the form of a
pharmaceutical
composition wherein the active compounds) is in admixture with one or more
3o pharmaceutically acceptable carriers, excipients or diluents.
Pharmaceutical compositions for
use in accordance with the present invention may be formulated in conventional
manner using
one or more physiologically acceptable Garners comprising excipients and
auxiliaries which
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facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
For injection, the agents of the invention may be formulated in aqueous
solutions,
preferably in physiologically compatible buffers such as Hanks's solution,
Ringer's solution,
or physiological saline buffer. For transmucosal administration, penetrants
appropriate to the
barrier to be permeated are used in the formulation. Such penetrants are
generally known in
the art.
For oral administration, the compounds can be formulated readily by combining
the
active compounds) with pharmaceutically acceptable Garners well known in the
art. Such
carriers enable the compounds of the invention to be formulated as tablets,
pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral
ingestion by a
subject to be treated. Pharmaceutical preparations for oral use can be
obtained solid excipient,
optionally grinding a resulting mixture, and processing the mixture of
granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable
excipients are, in
~ 5 particular, fillers such as sugars, including lactose, sucrose, mannitol,
or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice starch,
potato starch,
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,
disintegrating agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic
acid or a salt
thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar
solutions may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable
organic solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or
2s dragee coatings for identification or to characterize different
combinations of active
compound doses.
Pharmaceutical preparations which can be used orally 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 filler such
as lactose, binders such as starches, and/or lubricants such as talc or
magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or suspended
in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In
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addition, stabilizers may be added. All formulations for oral. administration
should be in
dosages suitable for such administration.
For buccal administration,the compositions may take the form of tablets or
lozenges
formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the dosage unit
may be determined
to by providing a valve to deliver a metered amount. Capsules and cartridges
of e.g. gelatin for
use in an inhaler or insufflator may be formulated containing a powder mix of
the compound
and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection,
e.g., by
bolus injection or continuous infusion. Formulations for injection may be
presented in unit
is dosage form, e.g., in ampoules or in mufti-dose containers, with an added
preservative. The
compositions may take such forms as suspensions, solutions or emulsions in
oily or aqueous
vehicles, and may contain formulatory agents such as suspending, stabilizing
and/or
dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions
20 of the active compounds in water-soluble form. Additionally, suspensions of
the active
compounds may be prepared as appropriate oily injection suspensions. Suitable
lipophilic
solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may
contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
25 cellulose, sorbitol, or dextran. Optionally, the suspension may also
contain suitable stabilizers
or agents which increase the solubility of the compounds to allow for the
preparation of highly
concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution
with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
3o The compounds may also be formulated in rectal compositions such as
suppositories
or retention enemas, e.g., containing conventional suppository bases such as
cocoa butter or
other glycerides.
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In addition to the formulations described previously, the compounds may also
be
formulated as a depot preparation. Such long acting formulations may be
administered by
implantation or transcutaneous delivery (for example subcutaneously or
intramuscularly),
intramuscular injection or a transdermal patch. Thus, for example, the
compounds may be
formulated with suitable polymeric or hydrophobic materials (for example as an
emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a
sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase
carriers or excipients. Examples of such Garners or excipients include but are
not limited to
to calcium carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin,
and polymers such as polyethylene glycols.
One product of the invention is a veterinary preparation of an aromatic
compound of
the invention, used alone or combined with an anti-scours agent. An anti-
scours agent is a
composition which is known to be useful in preventing or inhibiting the
symptoms of scours.
Known compositions include, for example, colostral extracts, such as those
described in U.S.
patent no. 4,377,569 and Canadian patent no. 1,175,352 and widely commercially
available
(e.g. Soluble Colostrum Powder, by VedCo, Inc., St. Joseph MO; Colostrum Bolus
II, by RX
Veterinary Frociu;,t~, Kar~a~ City ivlU, etc.); an immunological preparation
of colostrum
isolated from milk-producing mammals which may have been immunized against
certain
2o diarrheal causing microorganisms, such as those described in U.S. patent
no. 4,834,974,
Australian patent no. 39340/89, Australian patent no. 52547/90, and German
patent no.
1,560,344; microorganism specific immunological preparations, including
microorganism
specific hybridoma-derived monoclonal antibodies such as those described in
Sherman et al.,
Irlfec ion and Imnpunitv, V. 42 (2), P. 653-658 (1983) and a bovine
immunoglobulin fraction
prepared from bovine plasma or clear bovine serum such as the fraction
described in U.S.
patent no. 3,984,539; oral rehydration fluids and/or replacement electrolyte
compositions
which are widely commercially available in the form of dry compositions or
liquid solutions
prepared for oral or intravenous administration (e.g. Electrolyte H, by Agri-
Pet Inc., Aubrey
TX; Electrolyte Powder 8x, by Phoenix Pharmaceutical Inc, St. Joseph MO;
Electrolyte
3o Solution Rx, by Lextron Inc., Greeley CO, ProLabs LTD, St. Joseph MO, and
VetTek Inc.,
Blue Springs MO; Calf Rehydrate, by Durvet Inc., Blue Springs MO, etc.) and
antibiotic
compositions which are commercially available (e.g. BIOSOL~ Liquid, by The
UpJohn
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Company Animal Health Division, Kalamazoo MI; AMOXI-BOL~, by SmithKline-
Beecham
Animal Health, Exton PA; 5-WAY CALF SCOUR BOLUSTM, by Agri Laboratories LTD,
St.
Joseph MO; 1-A-DAY CALF SCOUR BOLUS, by A.H.A.; GARACIN~ PIG PUMP, by
Schering-Plough Animal Health Corporation, Kenilworth NJ, etc.).
In one embodiment, the veterinary preparation is a dry preparation of the
aromatic
compound of the invention and an antiscours agent. The dry preparation may be
administered
directly or may be hydrated and/or diluted in a liquid solution prior to
administration. In
another embodiment, the veterinary preparation is a liquid solution of the
compound of the
invention and an anti-scours agent.
1o Another product of the invention is a pharmaceutical preparation of an
aromatic
compound of the invention and an anti-diarrheal agent. An anti-diarrheal agent
includes, for
example, an immunoglobulin preparation from bovine colostrum; lomotil; an
intravenous or
oral rehydration fluid; a dry rehydration composition salt; an electrolyte
replacement
composition (in dry or liquid form); an oral or intravenous sugar-electrolyte
solution or dry
I5 composition; an antibiotic such as tetracycline, trirmethoprim or
sulfamethoxazole; a
quinolone drug such as norfloxacin or ciprofloxacin, bismuth subsalicylate,
diphenoxylate;
and loperamide.
In one embodiment the pharm~~~ueical preparation is a dry preparation of the
aromatic
compound of r~;;, ;"vention and an anti-diarrhea u~~;,l~, ~1'he dry
preparation may be
2o administered directly or may be hydrated and/or diluted in a liquid
solution prior to
administration. In another embodiment the pharmaceutical preparation is a
liquid solution of
the aromatic compound of the invention and an anti-diarrheal agent.
The time of administration of the aromatic compounds useful according to the
invention varies depending upon the purpose of the administration. When the
compounds of
25 the invention are administered in order to prevent the development of
diarrhea in subjects
traveling to areas with high risk of exposure to infectious agent or subjects
otherwise exposed
to diarrhea causing agents, the compounds should be administered at about the
time that the
subject is exposed to the risk or the high risk area. When the compounds are
administered to
subjects in order to prevent the development of scours, the veterinary
preparation should be
3o administered within the first 12 hours after birth, and preferably within
the first 4 hours after
birth. When the compounds of the invention are used to treat subjects having
symptoms of
diarrhea or scours, the compounds may be administered at any point while the
subject is
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experiencing symptoms, and preferably as soon as the symptoms develop. Other
considerations will be apparent when the compounds are used to treat
inflammatory diseases,
proliferative diseases, etc.
When administered, the formulations of the invention are applied in
pharmaceutically
acceptable amounts and in pharmaceutically acceptable compositions. Such
preparations may
routinely contain salts, buffering agents, preservatives, compatible carriers,
and optionally
other therapeutic ingredients. When used in medicine the salts should be
pharmaceutically
acceptable, but non-pharmaceutically acceptable salts may conveniently be used
to prepare
pharmaceutically acceptable salts thereof and are not excluded from the scope
of the
1 o invention. Such pharmacologically and pharmaceutically acceptable salts
include, but are not
limited to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric,
nitric, phosphoric, malefic, acetic, salicylic, p-toluene sulfonic, tartaric,
citric, methane
sulfonic, formic, maIonic, succinic, naphthalene-2-sulfonic, and benzene
sulfonic. Also,
pharmaceutically acceptable salts can be prepared as alkaline metal or
alkaline earth salts,
such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% WN); citric
acid and a
salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and
a salt
(0.8-2% W/V).
Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V);
2o chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal
(0.004-0.02%
W/V).
The active compounds of the present invention may be pharmaceutical
compositions
having a therapeutically effective amount of an aromatic compound of the
general formula
provided above in combination with a non-formula (I) active agent, optionally
included in a
pharmaceutically-acceptable carrier. The active compounds of the present
invention also may
be veterinary compositions having a therapeutically effective amount of an
aromatic
compound of the general formula provided above in combination with a non-
formula (I)
active agent, optionally included in a pharmaceutically-acceptable Garner. The
term
"pharmaceutically-acceptable carrier" as used herein means one or more
compatible solid or
liquid filler, dilutants or encapsulating substances which are suitable for
administration to a
human or other animal. The term "carrier" denotes an organic or inorganic
ingredient, natural
or synthetic, with which the active ingredient is combined to facilitate the
application. The
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components of the pharmaceutical compositions also are capable of being
commingled with
the compound of the present invention, and with each other, in a manner such
that there is no
interaction which would substantially impair the desired pharmaceutical
efficacy.
A common administration vehicle (e.g., pill, tablet, bolus, powder or solution
for
s dilution, pig pump, implant, injectable solution, etc.) would contain both
the compounds
useful in this invention and a non-formula (I) active agent. Thus, the present
invention
provides pharmaceutical or veterinary compositions, for medical or veterinary
use, which
comprise the active compounds of the invention together with one or more
pharmaceutically
acceptable carriers thereof and other therapeutic ingredients.
1o A variety of administration routes are available. The particular mode
selected will
depend of course, upon the particular drug selected, the severity of the
condition being treated
and the dosage required for therapeutic efficacy. The methods of this
invention, generally
speaking, may be practiced using any mode of administration that is medically
acceptable,
meaning any mode that produces effective levels of the active compounds
without causing
15 clinically unacceptable adverse effects. Such modes of administration
include oral, rectal,
topical, nasal, transdermal or parenteral routes. The term "parenteral"
includes subcutaneous,
intravenous, intramuscular, or infusion. Intravenous and intramuscular routes
are not
particularly suited for long term therapy and prophylaxis. They could,
however, be preferred
in emergency situations. Oral administration will be preferred for
prophylactic treatment
2o because of the convenience to the subject as well as the dosing schedule.
The compositions may conveniently be presented in unit dosage form and may be
prepared by any of the methods well known in the art of pharmacy. All methods
include the
step of bringing the active compounds into association with a carrier which
constitutes one or
more accessory ingredients. In general, the compositions are prepared by
uniformly and
25 intimately bringing the active compounds into association with a liquid
carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of the active compound, which is preferably isotonic with
the blood of
the recipient. This aqueous preparation may be formulated according to known
methods
3o using those suitable dispersing or wetting agents and suspending agents.
The sterile injectable
preparation may also be a sterile injectable solution or suspension in a non-
toxic
parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-
butane diol.
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Among the acceptable vehicles and solvents that may be employed are water,
Ringer's
solution, and isotonic sodium chloride solution. In addition, sterile, fixed
oils are
conventionally employed as a solvent or suspending medium. For this purpose
any bland
fixed oil may be employed including synthetic mono or di-glycerides. In
addition, fatty acids
such as oleic acid find use in the preparation of injectables. Carrier
formulations suitable for
oral, subcutaneous, intravenous, intramuscular, etc. can be found in
Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
Other delivery systems can include time-release, delayed release or sustained
release
delivery systems. Such systems can avoid repeated administrations of the
active compounds
of the invention, increasing convenience to the subject and the physician.
Many types of
release delivery systems are available and known to those of ordinary skill in
the art. They
include polymer based systems such as polylactic and polyglycolic acid,
polyanhydrides and
polycaprolactone; nonpolymer systems that are lipids including sterols such as
cholesterol,
cholesterol esters and fatty acids or neutral fats such as mono-, di- and
triglycerides; hydrogel
release systems; silastic systems; peptide based systems; wax coatings,
compressed tablets
using conventional binders and excipients, partially fused implants and the
like. Specific
examples include, but are not limited to: (a) erosional systems in which an
agent of the
invention is contained in a form within a matrix such as those described in
U.S. Patent Nos.
4,452,775, 4,675,189, and 5,736,152, and (b) diffusi~;,di systems in which an
active
2o component permeates ~~ a controlled rate from a polymer such as described
in U.S. Patent
Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware
delivery
systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for
treatment
of diarrhea in immunodeficient subjects, who need continuous administration of
the
compositions of the invention. "Long-term" release, as used herein, means that
the implant is
constructed and arranged to deliver therapeutic levels of the active
ingredient for at least 30
days, and preferably 60 days. Long-team sustained release implants are well
known to those
of ordinary skill in the art and include some of the release systems described
above.
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Pharmaceutical compositions suitable for use with the present invention
include
compositions wherein the active ingredient is contained in a therapeutically
effective amount,
i.e., in an amount effective to achieve its intended purpose. Of course, the
actual amount
effective for a particular application will depend, inter alia, on the
condition being treated. For
example, when administered in methods to reduce sickle cell dehydration and/or
delay the
occurrence of erythrocyte sickling or distortion in situ, such compositions
will contain an
amount of active ingredient effective to achieve this result. When
administered in methods to
inhibit cell proliferation, such compositions will contain an amount of active
ingredient
1o effective to achieve this result. When administered to subjects suffering
from disorders
characterized by abnormal cell proliferation, such compositions will contain
an amount of
active ingredient effective to, inter alia, prevent the development of or
alleviate the existing
symptoms of, or prolong the survival of, the subject being treated. For use in
the treatment of
cancer, a therapeutically effective amount further includes that amount of
compound which
arrests or regresses the growth of a tumor. Determination of an effective
amount is well
within the capabilities of those skilled in the art, especially in light of
the detailed disclosure
herein.
For any compound described herein the therapeutically effective amount can be
initially determined from cell culture assays. Target plasma concentrations
will be those
2o concentrations of active compounds) that ,;.iG capable of inducing at least
about 25%
inhidi~iu~: ;.~ ~i~e Uardos channel, at Ieast about 25% inhibition of CY
secretion in intestinal
cells and/or at least about 25% inhibition of cell proliferation in cell
culture assays,
depending, of course, on the particular desired application. Target plasma
concentrations of
active compounds) that are capable of inducing at least about 50%, 75%, or
even 90% or
higher inhibition of the Gardos channel, of Cf secretion in intestinal cells,
and/or of cell
proliferation in cell culture assays are preferred. The percentage inhibition
of the Gardos
channel, of Cf secretion in intestinal cells, and/or cell proliferation in the
subject can be
monitored to assess the appropriateness of the plasma drug concentration
achieved, and the
dosage can be adjusted upwards or downwards to achieve the desired percentage
of inhibition.
3o Therapeutically effective amounts for use in humans can also be determined
from
animal models. For example, a dose for humans can be formulated to achieve a
circulating
concentration that has been found to be effective in animals. Useful animal
models for
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diseases characterized by abnormal cell proliferation are well-known in the
art. A particularly
useful animal model for sickle cell disease is the SAD mouse model (Trudel et
al., 1991,
EMBO J.J. x:3157-3165). Useful animal models for diseases characterized by
abnormal cell
proliferation are well-known in the art. In particular, the following
references provide suitable
animal models for cancer xenografts (Corbett et al., 1996, J. Exn. Ther Oncol
] :95-108;
Dykes et al., 1992, Cont_rib Oncol Bas .1 Karg~ 42:1-22), restenosis (Carter
et al., 1994, ~
Am. Coll. Cardiol. 24(5):1398-1405), atherosclerosis (Zhu et al., 1994, rdiolo
v i~5(6):370-
377) and neovascularization (Epstein et al., 1987, rn a ø(4):250-257). The
dosage in
humans can be adjusted by monitoring Gardos channel inhibition and/or
inhibition of cell
proliferation and adjusting the dosage upwards or downwards, as described
above.
Additional in vivo assays are well known in the art. For instance, the
following assays
are useful for assessing effective amounts of compounds for treating
inflammatory diseases
associated with cellular proliferation: Airway inflammation and
hyperresponsiveness in
Ovalbunun-sensitized mice or guinea pigs; NZB/NZW crossed mice develop
glomerular
is disease and lupus-like syndrome; Renal allograft reiert~.~~, in mice;
Trinitrobenzene sulphonic
acid induced bowel inflamm_at:;.i~ in rats; NZB/NZW crosse : ..ice develop
glomerular disease
and lupus-like syndrome; Experimental allergic encephalomyelitis; Rat adjuvant
arthritis
assay; HLA transgenic mice immunized with thyroglobulin; and Thiouracil-fed
rats.
A therapeutically effective dose can also be determined from human data for
2o compounds which are known to exhibit similar pharmacological activities,
such as
Clotrimazole and other antimycotic agents (see, e.g., Brugnara et al., 1995,
JPET 2~3:266-
272; Benzaquen et al., 1995, Nature Medicine ,1_:534-540; Brugnara et al.,
1996, J. Clin.
Invest. Q~(S):1227-1234). The applied dose can be adjusted based on the
relative
bioavailability and potency of the administered compound as compared with
Clotrimazole.
25 Adjusting the dose to achieve maximal efficacy in humans based on the
methods
described above and other methods as are well-known in the art is well within
the capabilities
of the ordinarily skilled artisan.
Of course, in the case of local administration, the systemic circulating
concentration of
administered compound will not be of particular importance. In such instances,
the compound
3o is administered so as to achieve a concentration at the local area
effective to achieve the
intended result.
For use in the prophylaxis and/or treatment of sickle cell disease, including
both
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chronic sickle cell episodes and acute sickle cell crisis, a circulating
concentration of
administered compound of about 0.001 ~M to 20 ~M is considered to be
effective, with about
0.1 ~.M to 5 ~M being preferred.
Subject doses for oral administration of the compounds described herein, which
is the
preferred mode of administration for prophylaxis and for treatment of chronic
sickle cell
episodes, typically range from about 80 mg/day to 16,000 mg/day, more
typically from about
800 mg/day to 8000 mg/day, and most typically from about 800 mg/day to 4000
mg/day.
Stated in terms of subject body weight, typical dosages range from about 1 to
200 mg/kg/day,
more typically from about 10 to 100 mg/kg/day, and most typically from about
10 to 50
mg/kg/day. Stated in terms of subject body surface areas, typical dosages
range from about
40 to 8000 mg/m2/day, more typically from about 400 to 4000 mg/m2/day, and
most typically
from about 400 to 2000 mglm2/day.
For use in the treatment of disorders characterized by abnormal cell
proliferation,
including cancer, arteriosclerosis and angiogenic conditions such as
restenosis, a circulating
concentration of administered compound of about 0.001 ~M to 20 p,M is
considered to be
effective, with about 0.1 ~.M to 5 ~M being preferred.
Subject doses for oral administration of the compounds described herein for
the
treatment or prevention of cell proliferative disorders typically range from
about 80 mg/day to
16,000 mg/day, more typically from about 800 mglday to 8000 mg/day, and most
typically
from about 800 mg/day to 4000 mg/day. Stated in terms of subject body weight,
typical
dosages range from about 1 to 200 mg/kg/day, more typically from about 10 to
100
mg/kg/day, and most typically from about 10 to 50 mg/kg/day. Stated in terms
of subject
body surface areas, typical dosages range from about 40 to 8000 mg/m2/day,
more typically
from about 400 to 4000 mg/mz/day, and most typically from about 400 to 2000
mg/m2/day.
For other modes of administration, dosage amount and interval can be adjusted
individually to provide plasma levels of the administered compound effective
for the
particular clinical indication being treated. For example, if acute sickle
crises are the most
dominant clinical manifestation, a compound according to the invention can be
administered
in relatively high concentrations multiple times per day. Alternatively, if
the subject exhibits
only periodic sickle cell crises on an infrequent or periodic or irregular
basis, it may be more
desirable to administer a compound of the invention at minimal effective
concentrations and
to use a less frequent regimen of administration. This will provide a
therapeutic regimen that
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is commensurate with the severity of the sickle cell disease state.
For use in the treatment of tumorigenic cancers, the compounds can be
administered
before, during or after surgical removal of the tumor. For example, the
compounds can be
administered to the tumor via injection into the tumor mass prior to surgery
in a single or
several doses. The tumor, or as much as possible of the tumor, may then be
removed
surgically. Further dosages of the drug at the tumor site can be applied post
removal.
Alternatively, surgical removal of as much as possible of the tumor can
precede
administration of the compounds at the tumor site.
Combined with the teachings provided herein, by choosing among the various
active
1o compounds and weighing factors such as potency, relative bioavaiiability,
subject body
weight, severity of adverse side-effects and preferred mode of administration,
an effective
prophylactic or therapeutic treatment regimen can be planned which does not
cause substantial
toxicity and yet is entirely effective to treat the clinical symptoms
demonstrated by the
particular subject. Of course, many factors are important in determining a
therapeutic
15 regimen suitable for a particular indication or subject. Severe indications
such as cancer may
warrant administration of higher dosages as compared with less severe
indications such as
sickle cell disease.
The formulations of the invention are also administered in effective amounts
when
treating diarrhea or scours. An effective amount is one sufficient to inhibit
or prevent diarrhea
20 or scours and is thus sufficient to inhibit the Cf secretion of intestinal
epithelial cells. An
amount which is sufficient to inhibit the CY secretion of intestinal
epithelial cells thereby
effectively decreases the secretory response, thereby resulting in a decrease
in diarrhea or
scours andlor the symptoms thereof. Effective amounts will depend, of course,
on the
particular condition being treated; the severity of the condition; individual
subject parameters
25 including age, physical condition, size and weight; concurrent treatment;
frequency of
treatment; and the mode of administration. These factors are well known to
those of ordinary
skill in the art and can be addressed with no more than routine
experimentation.
An effective amount for an individual compound may be assesed using any method
known in the art which reliably determines the amount of Cl- secretion from
intestinal cells.
3o A compound may be subject to a series of standard assays or screens to
determine its
pharmacological activity and effective amounts.
In general, the active compounds of the invention are those which induce at
least about
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25% inhibition of the Cl- secretion, as measured using in vitro assays that
are commonly
known in the art (see, e.g., Example 4). Alternatively, or in addition, the
active compounds of
the invention generally will have an ICS° (concentration of compound
that yields 50%
inhibition) for inhibition of the CY secretion of less than about 10 ~,M as
measured using in
vitro assays.
It is preferred generally that a maximum dose be used, that is, the highest
safe dose
according to sound medical judgment, particularly if acute diarrhea or scours
are the dominant
clinical manifestation. Dosage may be adjusted appropriately to achieve
desired drug plasma
levels. Generally, daily oral doses of active compounds will be from about
0.01
1o milligrams/kg per day to 1000 milligrams/kg per day. It is expected that
oral doses in the
range of 50 to 500 milligrams/kg, in one or several administrations per day,
will yield the
desired results. In the event that the response in a subject is insufficient
at such doses, even
higher doses (or effective higher doses by a different, more localized
delivery route) may be
employed to the extent that subject tolerance permits. Multiple doses per day
are
15 contemplated to achieve appropriate systemic levels of compounds.
Toxicity
The ratio between toxicity and therapeutic effect for a particular compound is
its
therapeutic index and can be expressed as the ratio between LDS° (the
amount of compound
lethal in 50% of the population) and EDso (the amount of compound effective in
SO% of the
2o population). Compounds which exhibit high therapeutic indices are
preferred. Therapeutic
index data obtained from cell culture assays and/or animal studies can be used
in formulating
a range of dosages for use in humans. The dosage of such compounds preferably
lies within a
range of plasma concentrations that include the EDS° with little or no
toxicity. The dosage
may vary within this range depending upon the dosage form employed and the
route of
25 administration utilized. The exact formulation, route of administration and
dosage can be
chosen by the individual physician in view of the subject's condition. (See
e.g. Fingl et al.,
1975, In: The Pha_nnacological Basis ofTherap mt;~~~ Ch. 1 pl).
The invention having been described, the following examples are intended to
illustrate,
not limit, the invention. Some of the following examples depict tests that are
employed to
3o determine the effects on C 1- secretion. Clotrimazole, which is outside of
the scope of the
present claims is used to exemplify how the compounds of the present invention
are tested for
certain conditions. The compounds of the present invention are structurally
distinct from the
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structure of clotrimazole. Nevertheless, the compounds of the present
invention act on
chloride secretion in the same manner as clotrimazole and, therefore, are
useful in the methods
and products of the present invention.
F~.am les
F~xa_mnle 1: Com o nd ,gym BPS .s
This Example demonstrates general methods for synthesizing the compounds of
the
invention, as well as preferred methods for synthesizing certain exemplary
compounds of the
invention. In all the reaction schemes described herein, suitable starting
materials are either
commercially available or readily obtainable using standard techniques of
organic synthesis.
Where necessary, suitable groups and schemes for protecting the various
functionalities are
well-known in the art, and can be found, for example, in Kocienski, Protectins
c~rrnmc~ Georg
Thieme Verlag, New York, 1994 and Greene & Wuts, Protective Groups in Ors nir
Chem'y~,~yr , John Wiley & Sons, New York, 1991.
15 In FIGS. 1 and 2, the various substituents are as defined for structure
(I).
1 l~ynthesis of 11-Arvl-5 6-dihydro 11 H dibe"zfh P~ azP ine~
This example provides a general method for synthesizing substituted 11-aryl-
5,6-
dihydro-11H-dibenz[b,e]azepine compounds according to the invention. A general
reaction
scheme is provided in FIG. 1. In FIG. 1, Rz-R,5 are as previously defined for
structural
2o formula (I).
Referring to FIG. 1, a mixture of an appropriately substituted 2-
aminobenzophenone
100 (1 equivalent), an appropriately substituted benzyl chloride 102 (1
equivalent), potassium
carbonate (2 equivalents) and sodium iodide (1 equivalent) in acetonitrile is
refluxed for 12
hours. The reaction mixture is cooled to room temperature and water added. The
mixture is
25 extracted with ethyl acetate. The combined ethyl acetate extracts are
washed with water then
dried over sodium sulfate. Evaporation of the solvent followed by column
chromatography
gives the substituted N-alkyl-2-aminobenzophenone derivative 104 in about 55-
80% yield.
The substituted N-alkyl-2-aminobenzophenone derivative 104 ( 1 equivalent) is
dissolved in a 3:1 mixture of tetrahydrofuran:methanol. Sodium borohydride (10
equivalents)
30 is slowly added and the reaction mixture is stirred at room temperature for
12 hours. The
reaction is quenched by adding 2 N aqueous hydrochloric acid solution. The
reaction mixture
is neutralized by adding 4 N aqueous sodium hydroxide solution and extracted
with ethyl
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acetate. The combined ethyl acetate extracts are dried over sodium sulfate.
Evaporation of
the solvent followed by column chromatography gives the substituted N-alkyl-2-
amino-
benzyl alcohol derivative 106 in about 40-60% yield.
A mixture of the substituted N-alkyl-2-amino-benzylalcohol derivative 106 (1
equivalent), phosphorous pentoxide (5 equivalents) and methanesulfonic acid (5
equivalents)
in dichloromethane is stirred at room temperature for 12 hours. The mixture is
neutralized by
adding aqueous sodium carbonate and then extracted with dichloromethane. The
organic
solution is dried over sodium sulfate. Evaporation of the solvent followed by
column
chromatography gives the substituted 11-aryl-5,6-dihydro-11H-
dibenz[b,e]azepine derivative
108 in about 45-70% yield.
The substituted 11-aryl-5,6-dihydro-I IH-dibenz[b,e]azepine derivative 108 (1
equivalent) is combined with potassium carbonate (3.5 equivalents} and alkyl
or acyl halide (3
equivalents) in acetonitrile and stirred at room temperature for two days.
Water is added and
the mixture is stirred for 15 min. at room temperature and extracted with
ethyl acetate.
~ 5 Evaporation of the solvent gives the crude product as an oil. Tituration
of the product from
ethanol followed by washing with hexane gives the pure N-substituted I I-aryl-
5,6-dihydro-
11H-dibenz[b,e]azepine product 110 as a white solid in 30-80% yield.
Alternatively, the substituted 11-aryl-5,6-dihydro-1 IH-dibenz[b,e]azepines
can be
synthesized from appropriate starting materials according to the methods
described in
2o Sasakura and Sugasawa, 1981, Heteroc,~ x:421-425.
I.2. Synthesis of 11-Arvl-11-substituted-5,6-dihy~o-dibenz[b.e]aze in nes
This example provides a general method for synthesizing 1 I-aryl-11-
substituted-5,6-
dihydro-dibenz[b,e]azepine compounds according to the invention. A general
reaction
25 scheme is provided in FIG. 2. In FIG. 2, R,-R,5 are as previously defined
for structural
formula (I).
Referring to FIG. 2, the substituted N-alkyl-2-aminobenzophenone derivative
104 is
prepared as described in Section 1.1, supra. To a solution (0.25 M) of an
approriate grignard
reagent in diethyl ether at -40°C is added a solution (0.1 M) of the
substituted N-alkyl-2-
3o aminobenzophenone derivative 104 in diethyl ether. The mixture is stirred
at -40°C for 30
min., warmed to room temperature and quenched with water. Extraction with
ethyl acetate
and evaporation of the solvent gives the crude alcohol product 112 as an oil.
The alcohol
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product 112 is purified by column chromatography to give the pure alcohol
product 112 as a
white solid in 85-90% yield.
Compound 112 { 1 equivalent) is dissolved in dichloromethane to 0.5-1.0 M.
Phosphorous pentoxide (4 equivalents) and methane sulfonic acid (4
equivalents) are added
and the mixture is stirred at room temperature for 2 hours. The reaction is
quenched with
saturated aqueous sodium bicarbonate and extracted with ethyl acetate.
Evaporation of the
solvent followed by column chromatography gives the 11-aryl-11-substituted-5,6-
dihydro-
dibenz[b,e]azepine 114 in about 90% yield.
The 11-aryl-11-substituted-5,6-dihydro-dibenz[b,e]azepine 114 can be converted
to
to the corresponding N-substituted-11-aryl-11-substituted-5,6-dihydro-
dibenz[b,e]azepine 116 as
described in Section 1.1, supra.
1.3. S,~thesis of N-Methoxycarbon~(2'-chlorop~~)-5.6-dihydro-11 H-
diben ~[b elazepine (Compound 91
A preferred method of synthesis of N-methoxycarbonyl -11-(2'-chlorophenyl)-5,6-
dihydro-11 H-dibenz[b,e]azepine (Compound 9) is as follows: A mixture of 0.3 g
(0.00098
mole) of 11-(2'-chlorophenyl)-5,6-dihydro-11H-dibenz[b,e]azepine, 1.08 g
(0.0078 mole) of
potassium carbonate and 1.54 g (0.016 mole) of methyl chloroformate in 10 mL
of
acetonitrile, was refluxed for 12 hours. The mixture was then allowed to cool
to room
temperature and stirred with 15 mL of water for 10 minutes. The reaction
mixture was
extracted with ethyl acetate (2 x 15 mL). The organic layer was dried over
magnesium
sulfate. Evaporation gave the crude product as a brown solid. Trituration of
the crude product
with ethanol and washing the obtained solid with hexane gave 0.172 g (48%
yield) of a white
solid having a melting point of 159-161 °C.
The product gave the following analytical data: NMR (CDC13): b 3.10 ppm (3H,
s,
OCH3); S 4.45 ppm ( 1 H, d, J=10 Hz, CHIN); b 5.48 ppm ( 1 H, s, CH); 8 5.82
ppm ( 1 H, d,
J=10 Hz, CHZN); 8 6.94 ppm (1H, m, aryl); S 7.10 ppm (4H, m, aryl); 8 7.28 ppm
{6H, m,
aryl); S 7.69 ppm ( 1 H, m, aryl).
1.4. Synthesis ofN-Phenox~carbon -11-phenyl-5.6-dihvdro-11H-dibenz(b.e)~,~ine
lCog~nound 147
A preferred method of synthesis of N-phenoxycarbonyl-11-phenyl-5,6-dihydro-11H-
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dibenz[b,e]azepine (Compound 14) is as follows: A mixture of 0.25 g (0.00092
mole) of 11-
phenyl-5,6-dihydro-11 H-dibenz[b,e]azepine, 0.318 g (0.0023 mole) of potassium
carbonate
and 0.318 g (0.002 mole) of phenyl chloroformate in 10 mL of acetonitrile, was
stirred at
room temperature for 2 days. The mixture was stirred with 15 mL of water for
15 minutes
and then extracted with ethyl acetate (2 x 35 mL). The organic layer was dried
over
magnesium sulfate. Evaporation gave the crude product as an oily material.
Trituration of the
obtained oil with ethanol then washing it with hexane gave 0.285 g {80% yield)
of a white
solid having a melting point of 155-165 °C.
The product gave the following analytical data: NMR (CDCl3): 8 4.46 ppm ( 1 H,
d,
J=7 Hz, CHZN); 8 5.28 ppm (1H, s, CH); S 5.69 ppm (1H, d, J =7 Hz, CHZN); 8
6.52 ppm
(2H, m, aryl); b 6.98 ppm {2H, m, aryl); b 7.14 - 7.42 ppm (13H, m, aryl); 8
7.58 (1H, m,
aryl).
1.5. Synthesis ofN-Phenoxvcarbonyl-11-f2'-chloroyhenyl)-5.6-dihydro-11H-
~ 5 dibe b.e]azey'ne (Compound 267
A preferred method of synthesis of N-phenoxycarbonyl-11-(2'-chlorophenyl)-5,6-
dihydro-11H-dibenz[b,e]azepine (Compound 26) is as follows: A mixture of 0.2 g
(0.00065
mole) of 11-(2'-chlorophenyl)-5,6-dihydro-11H-dibenz[b,e]azepine, 0.18 g
(0.0013 mole) of
potassium carbonate and 0.204 g (0.0013 mole) of phenyl chloroformate in 10 mL
of
2o acetonitrile, was stirred at room temperature for 2 days. The mixture was
stirred with 15 mL
of water for 10 minutes and then extracted with ethyl acetate (2 x 35 mL). The
organic layer
was dried over magnesium sulfate, f Itered and the solvent was evaporated.
Trituration of the
obtained residue with ethanol then washing it with hexane gave 0.082 g (30 %
yield) of a
white solid having a melting point of 95-99°C.
25 The product gave the following analytical data: NMR (CDCl3): 8 4.50 ppm
(1H, d,
J=7 Hz, CHZN); 8 5.5 8 ppm ( 1 H, s, CH); 8 5.80 ppm ( 1 H, d, J=7 Hz, CHZN);
8 6.52 ppm (2H,
m, aryl); S 7.06 - 7.3 8 ppm ( 14H, m, aryl); 8 7.79 ppm ( 1 H, m, aryl).
30 1.6. Synthesis of N-(4'-Nitrobenzo~)-11-f '-chlo~Q ,~henXll-5.6-dihydro-11H-
diber~z[b,P~]azepine lComnound 287
A preferred method of synthesis of N-(4'-nitrobenzoyl)-11-(2'-chlorophenyl)-
5,6-
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dihydro-11H-dibenz[b,e]azepine (Compound 28) is as follows: A mixture of 0.2 g
(0.00065
mole) of 11-(2'-chlorophenyl)-5,6-dihydro-11 H-dibenz[b,e]azepine, 0.179 g
(0.0013 mole) of
potassium carbonate and 0.133 g {0.00072 mole) of 4-nitrobenzoyl chloride in
10 mL of
acetonitrile, was stirred at room temperature for 12 hours. The mixture was
stirred with 15
mL of water for 10 minutes. The reaction mixture was extracted with ethyl
acetate {2 x 15
mL). The organic layer was dried over magnesium sulfate. Evaporation gave the
crude
product as a sticky solid. Trituration of the crude product with ethanol and
washing the
obtained solid with hexane gave 0.148 g (50% yield) of a white solid having a
melting point
of 178-181 °C.
1o The product gave the following analytical data: NMR (CDCl3): 8 4.42 ppm
(1H, d,
J=7 Hz, CHZN); 8 5.68 ppm (1H, s, CH); 8 6.36 ppm (1H, d, J=7 Hz, CHZN); S
6.52 ppm (3H,
m, aryl); 8 7.06 ppm (3H, m, aryl); 8 7.12 ppm (1H, m, aryl); b 7.26 ppm (6H,
m, aryl); 8
7.79 ppm (3H, m, aryl).
~ 5 1.7. Other Com on unds
Other compounds of the invention can be synthesized by routine modification of
the
above-described syntheses, or by other methods that are well known in the art.
Appropriate
starting materials are commercially available or can be synthesized using
routine methods.
20 E~~g 2: In Yitro Activirir
This Example demonstrates the ability of several exemplary compounds of
formula (I)
to inhibit the Gardos channel of erythrocytes (Gardos channel assay) and/or
mitogen-induced
cell proliferation (mitogenic assay) in vitro. The assays are generally
applicable for
demonstrating the in vitro activity of other compounds of formula (I).
25 Methods. The percent inhibition of the Gardos channel (10 ~M compound) and
the
ICS° were determined as described in Brugnara et al., 1993, J. Biol.
Chem. ~$(12):8760-
8768. The percent inhibition of mitogen-induced cell proliferation (10 ~tM
compound) and
the ICS° were determined as described in Benzaquen et al. (1995, Nature
Medicine ~:534-
540) with NIH 3T3 mouse fibroblast cells (ATCC No. CRL 1658). Other cell
lines, e.g.,
3o cancer cells, endothelial cells and fibroblasts, as well as many others,
may be used in the cell
proliferation assay. Selection of a particular cell line will depend in part
on the desired
application, and is well within the capabilities of an ordinarily skilled
artisan.
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Resu~s_. The results of the experiment are provided in Table 2, below.
Clotrimazole is
reported for purposes of comparison. Most of the compounds tested exhibited
significant
activity in both assays. All of the compounds tested exhibited significant
activity in at Ieast
one of the assays.
Table Z
IN VITRO DATA FOR EXEMPLARY COMPOUNDS
Mitogenic Gardos Channel
Assay Assay
l0 Compound ICso Inhibition ICS Inhibition
(p,M) (%) (wM) (%)
Clotrimazole 0.626 93.0 0.046 99.3
(1) 56.0 0.775 75.2
(2) 5.20 99.0 1.30 99.0
(3) 2.40 99.0 0.886 97.4
15 (4) 1.5 89.0 0.384 98.1
(S~ 91.0 > 10.0 14.4
(6) 87.0 0.236 97.5
(7) I .60 99.0 > 10.0 35.8
(8) 2.20 84.0 0
20 (9) 2.10 99.0 0.0850-0.093 97.3
(10) 53.0 1.533-1.940 63.0
(11) 32.0 >10.0 9.5
(12) 13.0 > 10.0 54.8
(13) 1.7 97.0 0
25 (14) 0.04 98.0 >10.0 14.8
(15) 40.0 > 10.0 9.50
(16) 1.7 99.0 > 10.0 0.45
(17) 1.6 99.0 > 10.0 20.6
(18) 2.6 99.0 0.502-0.692 81.5
30 (19) 1.6 99.0 >10.0 52.0
(20) 1.7 95 .0 > 10.0 13.6
(21 ) 2.7 93 .0 > 10.0 2.1
(22) 3.6 99.0 > 10.0 14.9
23 55.0 > 10.0 18.2
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Mitogenic ~ Gardos Channel
Assay Assay
1o Compound ICso Inhibition ICso Inhibition
(~uM) (%) (wM) (%)
(24) 89.0 > 10.0 32-55
(25) 75.0 > 10.0 8.5
(26) 0.04-0.90 99.0 > 10.0 0.8
(27) 2.20 99.0 > 10.0 3.0
(28) 0.04-0.50 99.0 0
(29) 0.800 99.0 0.414-0.433 95.1
(30) 0.600 99.0 > 10 14.6
(31 ) 0.400 99.0 > 10 12.3
(32) 1.100 99.0 0
(33) 2.400 99.0 > 10 67.5
(34) 4.00 99.0 > 10 I 2.0
(35) 0 0.071-0.099 98.3
Fple 3: Activitv In Cancer Cell Lines
This Example demonstrates the antiproliferative effect of several exemplary
compounds of formula (I) against a variety of cancer cell lines. The assays
are generally
applicable for demonstrating the antiproliferative activity of other compounds
of formula (I).
ethods. Growth o Cells. The antiproliferative assays described herein were
performed using standard aseptic procedures and universal precautions for the
use of tissues.
2o Cells were propagated using RPMI 1640 media (Gibco) containing 2% or 5%
fetal calf serum
(FCS) (Biowhittaker) at 37°C, 5% COZ and 95% humidity. The cells were
passaged using
Trypsin (Gibco). Prior to addition of test compound, the cells were harvested,
the cell number
counted and seeded at 10,000 cells/well in 100 pl 5% fetal calf serum (FCS)
containing RPMI
medium in 96-well plates and incubated overnight at 37°C, 5% COz and
95% humidity.
On the day of the treatment, stock solutions of the test compounds (10 mM
compound/DMSO) were added in 100 pl FCS containing medium to a final
concentration of
10-0.125 pM and the cells were incubated for 2, 3 or 5 days at 37°C, 5%
COZ and 95%
humidity.
Following incubation, the cellular protein was determined with the
Sulforhodamine B
(SRB) assay (Skehan et al., 1990, ~,, Natl. Cancer Inst. $x:1107-1112). Growth
inhibition,
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reported as the concentration of test compound which inhibited SO% of cell
proliferation
(ICSo) was determined by curve fitting.
Values for VP-16, a standard anti-cancer agent, are provided for comparison.
Except for MMRU cells, all cancer cell lines tested were obtained from the
American
Type Culture Collection (ATCC, Rockville, MD). The ATCC assession numbers were
as
follows: HeLa (CCL-2); CaSki (CRL-1550); MDA-MB-231 (HTB-26); MCF-7 (HTB-22);
A549 (CCL-185); HTB-174 (HTB-174); HEPG2 (HB-8065); DU-145 (HTB-81); SK-MEL-
28 (HTB-72); HT-29 (HTB-38); HCT-15 (CCL-225); ACHN (CRL-1611); U-118MG (HTB-
15); SK-OV-3 (HTB-77).
to MMRU cells (Stender et al., 1993, 3. Dermatolo~v ~Q:611-617) were a gift of
one of
the authors.
Results. The results of the cell culture assays are presented in Tables 3 and
4, below.
CA 02310773 2000-OS-19
WO 99I26b28 _ S~ _ PCTIUS98/24967
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CA 02310773 2000-OS-19
WO 99/26628 _ 5g _ PCTIUS98/249b7
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0 RS CO b~ ld l~ G~ ~ l~ CtjGd C~ l'~GC~~ CCjC~
~
\ 'C 'C T7 "C 'O 'C 'C "O 'Ci'C T3 'O 'C "O 'CJ'O
~
M M N N N N M M M N M M M M N N
V
O o 0 0 0 0 0 0 0 0 0 o a o 0 0 0
U N N ~1 V1 v1 ~ mn ~n v~ ~n v~ v~ ~n ~ v~ ~n
\
0
G
O
G ~' V7 ~D ~ ~ ~D t~ 00 Ov O v~ N M ~ 1n
N N N N M M t~ tr1M M
O
U
~n o v~ o
-i .-~ N
CA 02310773 2000-OS-19
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As can be seen in Tables 3 and 4, compounds which exhibited significant
activity in
the mitogenic assay described in Section 7, supra, exhibit significant
antiproliferative activity
against a variety of cancer cell lines and cancer types (ICSO of less than
about 10 pM).
Many of the compounds exhibit comparable or even greater antiproliferative
activity
against a variety of cancer cell types than VP-16, a known anti-cancer agent.
Example 4: Formuja ions
The following examples provide exemplary, not limiting, formulations for
administering the compounds of the invention to mammalian, especially human,
patients.
i o Any of the compounds described herein, or pharmaceutical salts or hydrates
thereof, may be
formulated as provided in the following examples.
4.1. tablet Formulation
Tablets each containing 60 mg of active ingredient are made up as follows:
~ 5 Active Compound 1 SO mg
Starch 1 SO mg
Microcrystalline Cellulose 150 mg
Sodium carboxymethyl starch 4.5 mg
Talc 1 mg
2o Polyvinylpyrrolidone ( 10% in 4 mg
water)
Magnesium Stearate 0.5 mQ
160 mg
25 The active ingredient, starch and cellulose are passed through aNo. 45 mesh
U.S.
sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with
the resultant
powders which are then passed through a No. 14 mesh U.S. sieve. The granules
are dried at
50°-60°C and passed through a No. 18 mesh U.S. sieve. The sodium
carboxymethyl starch,
magnesium stearate and talc, previously passed through a No. 60 mesh U.S.
sieve, are then
3o added to the granules, which, after mixing are compressed by a tablet
machine to yield tablets
each weighing 150 mg.
Tablets can be prepared from the ingredients listed by wet granulation
followed by
compression.
CA 02310773 2000-OS-19
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4.2. Gelatin Ca sn ules
Hard gelatin capsules are prepared using the following ingredients:
Active Compound 250 mg/capsule
Starch dried 200 mg/capsule
Magnesium Stearate 10 mg/capsule
The above ingredients are mixed and filled into hard gelatin capsules in 460
mg
quantities.
to 4.3. Aerosol Solution
An aerosol solution is prepared containing the following components:
Active Compound 0.25% (w/w)
Ethanol 29.75% (w/w)
Propellant 22 77.00% (w/w)
(Chlorodifluoromethane)
The active compound is mixed with ethanol and the mixture added to a portion
of the
propellant 22, cooled to -30°C and transferred to a filling device. The
required amount is then
2o fed to a stainless steel container and diluted with the remainder of the
propellant. The valve
units are then fitted to the container.
4.4. S~positories
Suppositories each containing 225 mg of active ingredient are made as follows:
Active Compound 225 mg
Saturated fatty acid glycerides 2,000 mg
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended
in the
3o saturated fatty acid glycerides previously melted using the minimum heat
necessary. The
mixture is then poured into a suppository mold of nominal 2 g capacity and
allowed to cool.
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4.5. Suspensions
Suspensions each containing 50 mg of medicament per 5 mL dose are made as
follows:
Active Compound 50 mg
Sodium carboxymethylcellulose 50 mg
Syrup 1.25 mL
Benzoic acid solution 0.10 mL
Flavor q.v.
Color q.v.
Purified water to 5 mL
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed
with the
sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic
acid
solution, flavor and some color are diluted with some of the water and added,
with stirring.
Sufficient water is then added to produce the required volume.
Example 5: Clotrimazole inhibits water and electrolyte se~~tion in intestinal
epithelial
cells.
The biochemical basis of secretory diarrhea involves intestinal CY secretion
in
intestinal crypt cells. Under normal conditions, Cl- ions are maintained
within intestinal crypt
cells at levels above their electrochemical potential by primarily and
secondarily active
transport mechanisms such as the Na/K ATPase pumps and NaIK/2Cl
cotransporters. Cl- is
transported into the lumen from the intestinal crypt cells through apical Cf
channels.
Intracellular levels of K+, cAMP, cGMP, and Ca++ are all involved in
regulating the secretory
response.
T84 cells were used to determine whether clotrimazole regulates CY secretion
in
intestinal crypt cells. T84 cells form confluent monolayers of columnar
epithelia that exhibit
high transepithelial resistances, polarized apical and basilateral membranes,
and CAMP and
3o Ca++ regulated Cl- secretory pathways analogous to those found in native
intestine.
Methods. Growth of T84 cells. T84 cells obtained from ATCC were cultured and
passaged in equal parts of dulbecco's modified eagle's medium (DMEM), 1 g/ 1 D-
glucose) and
Hams F-12 nutrient mixture, supplemented with 5% newborn calf serum, 15 mM
HEPES, 14
mM Na HC03, 40mg11 penicillin, 8mg/1 ampicillin, 0.90 mg/1 streptomycin. Cells
were
seeded at confluent density onto 0.33 cmz or Scmz Transwell inserts (Costar,
Cambridge, MA)
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coated with dilute rat collagen solution as previously described (Lencer et
al., J. Clin. Invest.,
92: 2941-2951 (1993); Lencer et al., J. Cell Biol. 117: 1197-1209 (I992).
Transepithelial
resistances attain stable levels (>1000 Ohms.cmZ) after 7 days. The
development of high
transepithelial resistances correlated with the formation of confluent
monolayers with well-
s developed tight junctions as assessed by morphological analysis, and with
the ability of
monolayers to secrete Cl- (Madam et al., tr 92: 1133-1145 (1987).
Electro~ysiologv (mesurement of electrogenic Cl- secretionl. Confluent
monolayers
were transferred to Hanks Buffered Salt Solution (HBSS) containing 0.185 g/1
CaCl2, 0.098
gll MgS04, 0.4 gll KCI, 0.06 gll KHzP04, 8 NaCI, 0.048 g/1 Na2HP0~, 1 g/1
glucose, and
1 OnM HEPES, pH 7.4. Serosal and mucosal reservoirs were interfaced with
Calomel and Ag-
Ag Cl electrodes via 5% agar bridges made with Ringer's buffer.
Transepithelial resistance
was measured using a dual voltage clamp device to apply 25 or SOpA current
pulses. Short
circuit current (ISC) was calculated using Ohms law as previously described
(Lencer et al., ~
Clin. Invest. 92: 2941-2951 (1993); Lencer et al. J. Cell Biol. 117: 1197-1209
(1992).
15 Results: Clotrimazole reversibly inhibits Cl- secretion elicited by Ca++-
or cAMP-
dependant agonists in T84 cells. Previous studies have shown that Cf secretion
in T84 cells
is controlled by K+ efflux pathways which are biophysically and
pharmacologically distinct
from one another. One pathway participates in the secretory response to cAMP-
dependent
agonists and displays sensitivity to Ba++ salts (McRoberts, et al., J. Biol.
Chem. 260: 14163-
20 14172 (1985); Reenstra, Am J. Physiol. 264: C161-168 (1993)). The other
mediates the
response to Ca++-dependent agonists, and is Ba++-insensitive. Several pathway
specific
agonists of K+ channels are useful for determining whether a particular
compound is
functioning through a cAMP or Ca++ specific pathway. For instance, vasoactive
intestinal
peptide (VIP) and cholera toxin are cAMP mediated agonists of the K+ channel,
whereas,
25 carbachol is a Ca++-dependent agonist of the Ca++ regulated K+ channels.
The pathway by
which a particular inhibitor of CY secretion in T84 cells is functioning may
be identified by
measuring the ability of the inhibitor to modify transepithelial resistances
in T84 cells which
have been treated with VIP or carbachol to stimulate Cl- secretion.
T84 cells were grown as described above and Cf secretion was stimulated by the
3o addition to the media of either carbachol (100mM) or VIP (5nM). The cells
were then treated
with BaCI (3mM), charybdotoxin (100nM), or clotrimazole (33mM). The short
circuit current
(ISC) was determined for the various inhibitor treatments as a percentage of
the control in the
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absence of inhibitor (FIG. 3). BaCI strongly inhibited the secretory response
to the cAMP
mediated agonist VIP, but had no apparent affect on the secretory response
elicited by the
Ca++-dependent agonist carbachol. In contrast, the scorpion venom
Charybdotoxin strongly
inhibited the secretory response elicited by carbachol, but had minimal
affects on CI' secretion
elicited by VIP. However, clotrimazole inhibited the Cf secretory responses to
both agonists.
Inhibition of Cl- secretion by clotrimazole was fully reversible (9612%, n =
4) after 60 min
recovery in the presence of 0.01 mg/ml bovine serum albumin.
To examine possible effects of clotrimazole on the synergy between CAMP and
Ca++-
mediated agonists, monolayers, initially stimulated with VIP were allowed to
reach steady-
1o state levels of secretion and then additionally exposed to carbachol (100
pM). Clotrimazole
was slightly more effective in inhibiting the secretory response to carbachol
than to cAMP
with IC50 values of 3 and 8 ~,M, respectively. When the effects of
clotrimazole on cAMP-
and Ca++-dependent secretory pathways were examined on the same monolayers.,
inhibition of
the synergistic response to VIP plus carbachol was found to parallel the
inhibition of secretion
~ 5 promoted by Ca++ agonists alone. In low doses (=10-' or less),
clotrimazole potentiated
slightly (by S-10%) the CI-secretory responses to either agonist. clotrimazole
inhibited
effectively the secretory response to cholera toxin (20 nM, a cAMP-dependent
agonist) and E
Coli heat-stabile toxin (100 nm, a cGMP-agonist) (IC50 values of 10 pM and 15
~.M,
respectively).
2o The effect of clotrimazole on K+ conductances was also examined by isotopic
flux
studies using $6RB. T84 cells were grown in the presence of a cAMP agonist,
VIP, or a Ca++
mediated agonist (Thapsigargin). Clotrimazole was added and g6RB efflux was
measured.
Clotrimazole significantly inhibited baseline and Ca++ stimulated g6RB efflux
in the presence
of both cAMP and Ca++ mediated agonists compared to those cells which were not
treated
z5 with clotrimazole.
Other aromatic compunds of the invention were found to inhibit chloride
secretion.
Although clotrimazole was the most potent inhibitor tested of cAMP and Ca++
elicited Cl
secretion, ketoconazole, econazole, miconazole, and 2-chlorophenyl-bis-phenyl
methanol also
were effective at inhibiting chloride secretion.
3o Taken together, these studies indicate that clotrimazole inhibits Cf
secretion elicited
by cAMP or Ca++ mediated K+ channels in T84 cells .
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Exa_n,__~e 6: Clotrimazole acts at distal steps in the cAMP and a++-dependent
signal
tranSd~rtinn ~~athways.
To determine the site of clotrimazole action, the effects of clotrimazole
pretreatment
were examined on monolayers stimulated with agonists that initiate Cl-
secretion at sequential
steps in the cAMP signalling cascade. T84 monolayers were preincubated in HBSS
in the
presence or absence of clotrimazole (33 ~.M) and then stimulated with either 5
~.M VIP
(which activates adenylate cyclase through heterotrimeric GTPase-linked cell
surface
receptors), 10 ~M forskolin (which activates adenylate cyclase directly), or 3
mM 8Br-cAMP
(a direct stimulator of protein kinase A). Clotrimazole inhibited the
secretory response to
to each of these agonists. These data provide evidence that clotrimazole acts
at a step distal to
the activation of Protein Kinase A.
Ca~+-dependent intracellular signaling in T84 and other non-exciteable cells
involves
recruitment of inositol trisphosphate (IP3)-dependent intracellular Ca++
stores (Halm and
Frizzell, Textbook of Secretory Diarrhea, Raven Press, 47-58 (1990); Mandel et
al., J. Biol.
Chem. 267: 704-712 (1986); Halm et al., Am. J. Physiol. (Cell Physiol. 23)
254:C505-C511
(1988)), and subsequent activation of plasma membrane Ca++ influx pathways
(Barrett, Am.
J. Physiol. (Cell Physiol. 34): C859-C868 (1993)). Downstream events may be
mediated by
[Ca++]i, IP3, diacylglycerol, or as yet unidentified diffusable factors
(Putney and Bird, Cell
75:199-201 (1993)). To examine the site of clotrimazole action alone, this
signalling,
2o cascade, T84 monolayers pretreated in the presence or absence of
clotrimazole (33 ~M) were
stimulated with the Ca++-dependent agonists carbachol ( 100 ~M which elicits
both Ca++ and
IP3 signals), thapsigargin (5 ~M, which elevates cytoplasmic Ca++ via
inhibition of ER Ca++-
ATPase) (Vandorpe et al., Biophys. .I. 66:46-58 (1994)), or the Ca++ ionophore
ionomycin
(10 ~M). Clotrimazole inhibited strongly the Cl-secretory response to each to
these reagents.
These data suggest that clotrimazole acts at steps in the secretory response
distal to the release
of intracellular Ca++ stores.
Example 7: Clotrimazole does not affect apical membrane anion conductance or
basolateral
Na-K2C1 cotra_n_sps er .
~thods: ''--SI Efflux Studies. Confluent monolayers on 5 cmz Transwell inserts
were
used 10-14 days after plating.'z5I was measured as an indicator of apical Cl-,
channel and
basolateral K+ channel activity as previously described (Venglarik, et al, Am.
J. Physiol. (Cell
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Physiol. 28):C358-C364 (1990). Monolayers were preincubated at 37° C
with 4 pCi/ml'z5I
in HBSS for 90 minutes, with 33 pM clotrimazole absent or present during the
final 30
minutes of this 90 min preincubation period. Clotrimazole pretreatment did not
alter'z5I
loading of the cells. After washing twice in fresh HBSS, 0.5 ml samples were
obtained every
two min from the apical reservoir and replaced with fresh HBSS. After four
baseline samples
were obtained, the cells were treated (at t = 8 minutes) with vasoactive
intestinal peptide (VIP,
5 pnM) or thapsigargin (5 p.M) to stimulate C1- secretion, and an additional
15 timed samples
were obtained. Finally, the cell monolayer was rinsed, cut with its support
from the
polystyrene ring, and the residual cell-associated radioactivity was
determined. Monolayers
I o were maintained at 37 ° C in room air throughout the study. 'z5I
was counted by gamma
counting and normalized to percent total uptake as previously described
(Venglarik, et al, Am.
J. Physiol. (Cell Physiol. 28):C358-C364 (1990).
86Rb Uptake Studies. Confluent monolayers on 5 cmz Transwell inserts were
incubated for 30 minutes in HBSS at 37° C. A group of control and CLT
treated (33 ~M, for
30 min) monolayers were treated with bumetanide (10 pM for 12 min). All
monolayers were
then treated with VIP (SnM and shifted to HBSS containing 1 p,Ci/ml 86Rb for 3
minutes at
37° C. 86Rb uptake was terminated by washing the inserts in an ice-cold
solution containing
100mM MgCIz, and iOmM TRIS-CL, pH 7.4. Monolayers were cut from their inserts,
placed
into scintillation vials, and counted using standard methods.
$gsStudies were conducted to determine whether the inhibition of electrogenic
CI- secretion might occur by blockade of apical membrane Cl-channels, or
blockade of
basolaterally situated NaK2C1 cotransporters. To determine if clotrimazole
affected ion
conductance through apical membrane Cl-channels, we examined the time course
of'z5I efflux
from T84 monolayers pretreated in the presence or absence of clotrimazole
(Venglarik, et al,
Am. J. Physiol. (Cell Physiol. 28):C358-C364 (1990). Clotrimazole had little
or no effect on
the time course of'z5I efflux from monolayers treated with VIP. Rate constants
for 125I
efflux from monolayers treated or not treated with clotrimazole were
indistinguishable
(0.0637 vsØ0645 % uptake/minute, n=2 in duplicate). Clotrimazole had similar
lack of effect
on'z5I efflux stimulated by thapsigargin.
3o We next tested the effect of clotrimazole on basolateral NaK2CI
cotransporters, as
assessed by bumetanide-sensitive 86Rb uptake (Matthews et al., J. Biol. Chem.
269:15703-
15709 (1994)). Clotrimazole treatment reduced the total amount of ~Rb uptake
by 53.65.8%
CA 02310773 2000-OS-19
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(mean+SEM. n=6), but had no effect on the fractional component that was
bumetanide-
sensitive (88+3.2 vs 75.2 +12.7% total uptake, mean+SEM). Taken together,
these data
strongly suggest that clotrimazole does not affect CI- secretion in T84 cells
via inhibition of
either apical membrane Cl- channels or basolateral membrane NaK2C1
cotransporters.
F~r,~nie 8: Clotrimazole in_h_ibits Chloride secretion by inhibiting K+ efflux
through
basolateral K+ channels in T84 cells.
8.1. Clotrimazole inhibits chloride secretion by blockade of K+ tra~nort
through both Ba++-
to sensitive and charybdotoxin-sensitive channels.
ethods: g6Rb Efflux Studies Confluent monolayers on 5 cm2 Transwell inserts
were
used 10-14 days after plating. 86Rb flux was measured as an indicator of
apical Cl-, channel
and basolateral K+ channel activity as previously described (Venglarik, et al,
Am. J. Physiol.
(Cell Physiol. 28):C358-C364 (1990). Monolayers were preincubated at
37° C with 4 ~Ci/ml
15 B6Rb in HBSS for 90 minutes, with 33 ~M clotrimazole absent or present
during the final 30
minutes of this 90 min preincubation period. clotrimazole pretreatment did not
alter 86Rb
loading of the cells. One ml samples were obtained and replaced from the
basolateral
reservoir. After four baseline samples were obtained, the cells were treated
(at t = 8 minutes)
with vasoactive intestinal peptide (VIP, 5 ~nM) or thapsigargin (5 ~M) to
stimulate Cl-
2o secretion, and an additional 15 timed samples were obtained. Finally, the
cell monolayer was
rinsed, cut with its support from the polystyrene ring, and the residual cell-
associated
radioactivity was determined. Monolayers were maintained at 37° C in
room air throughout
the study. 86Rb was counted by scintillation counting and normalized to
percent total uptake
as previously described (Venglarik, et al, Am. J. Physiol. (Cell Physiol.
28):C358-C364
25 ( 1990).
Results: K+ channel activity was estimated by measurement of g6Rb efflux.
Clotrimazole was found to significantly inhibit the rate of 86Rb efflux after
treatment with the
cAMP agonist VIP (S ~tM). The rate constant For VIP-stimulated g6Rb efflux was
reduced by
87% in monolayers treated with clotrimazole (0.0062 vs. 0.0465 %
uptake/minute, n=2 in
3o triplicate). clotrimazole inhibited to a similar degree 86Rb efflux from
monolayers stimulated
with thapsigargin (panel B, rate constants 0.011 vs. 0.048% uptakelminute,
n=2), suggesting
that clotrimazole can inhibit Cl- secretion by blockade of K+ transport
through both Ba++-
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sensitive and charybdotoxin-sensitive channels.
8 2 Clotrimazole inhibits chloride secretion through distinct cAMP and Ca'+
sensitive
~olateral K+ channels.
Methods: Selective membrane Permeabilization and Measurement of Potassium
Conductance of the Basolateral Membrane. The basolateral potassium conductance
was
measured using the technique developed by Dawson and co-workers. A potassium
gradient
(mucosal to serosal) was first established across the monolayer using
asymmetric mucosal and
serosal buffers containing K+ as the sole permeant ion. The addition of
amphotericin B (20
~M) to the mucosal reservoir forms conductive pores in the apical membrane,
and thus
removes all resistance to transepithelial potassium movement across this
membrane. Thus,
under the conditions of the experiment, in which the monolayer is short
circuited (i.e.,
voltage-clamped at zero potential) and the transepithehal potassium gradient
is constant, the
amphotericin-dependent Isc becomes a measure of the rate of the
transepithelial potassium
flux across basolateral membranes. Changes in short circuit current (Isc),
then represent
changes in basolateral K+ conductances (gK). Isc and K+ conductances were
measured using
calomel electrodes, 3M KCI-agar bridges, and a voltage clamp (University of
Iowa, Iowa
City). To generate a voltage-current channel relationships, currents were
elicited by 1 sec test
potentials from -80 to +80 in 10 mV increments in the asymmetrical high K+
gluconate
solution.
Calculation of basolateral membrane K+ permeability. Membrane permeabilities
were
calculated according to the formula:
PK= (cm/s)='K (mM/cm2~s)/0[ K+] (mM/cm3)
where 0 [K+] is equal to the difference in K+ concentration (135 mM) between
the asymmetric
apical and basolateral bathing solutions. Maximal Isc values were converted
into K+ fluxes by
dividing by the Faraday constant F (96,500 coulombs/mol) as previously
described (Huflejt et
al., J. Clin. Invest. 93: 1900-1910 (1994)).
Results Basolateral K+ transport was examined in T84 monolayers perrneabilized
apically by pretreatment with amphotericin B. Apical and basolateral buffers
contained K+ as
3o the sole permeant ion. All studies were performed with a 135 mM
basolaterally directed K+
gradient. This method has been utilized previously to examine both Cl- and K+
transport in
T84 cells and HT29-C1.16E cells. Briefly, ion conductances in the luminal or
basolateral
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membranes of confluent T84 cell monolayers can be assessed separately by
selectively
permeabilizing the apical or basolateral membrane using the ionophore
amphotericin B. This
artificially removes all electrical resistance to ion transport across the
plasma membrane
containing pores formed by arnphotericin B. As a result, the intact
contralateral plasma
membrane becomes rate limiting for transepithelial ion transport. Agonist-
dependent changes
in ion conductances can be assessed directly either as transepithelial short
circuit current (Isc)
in the presence of established ion gradients, or as transepithelial
conductance (G) in the
presence of established transepithelial potentials.
K+ transport was measured at baseline and after the ordered additions of cAMP-
and
1 o Ca++ -agonists. The initial permeabilization with amphotericin B was
associated with 49 t
19% increase in conductance. Pores formed by amphotericin B display
selectivity for
monovalent cations. Ca++ remained relatively impermeant as evidenced by the
small steady
state increase in Isc and GK caused by apical permeabilization with
amphotericin B. Given
this low baseline Isc and GK, both cAMP- and Ca++-sensitive K+ permeabilities
(PK) were
readily apparent after agonist stimulation. Treatment with the cAMP-agonist
forskolin (10
~eM) caused a brisk increase in K+ transport through apparently low-
conductance pathway(s),
as evidenced by symmetrical increases in Isc and G. Carbachol also increased
K+ currents.
The magnitude of the carbachol-induced IscK, however, was similar whether
carbachol was
added alone or after forskolin (111.7 ~ 7.4 vs. 180.7 ~ 15.7~A/cm2
respectively. Thus, there
2o was no clear evidence of synergy between cAMP and Ca++ mediated K+
pathways, as would
be expected in an apically permeabilized cell system. Analagous to our
previous findings in
intact T84 monolayers, the forskolin-induced changes in Isc were sustained
while the effect of
carbachol was short-lived. Both IscK and GK returned to baseline values within
5 min after
addition of carbachol.
Formal currentlvoltage (I/V) relations were defined before and after agonist
stimulation to confirm that both cAMP- and Ca++-dependent currents were
elicited at
physiologic membrane potentials. Thapsigargin was used in place of carbacol as
a Ca++-
agonist in these studies because the K+ transients elicited by thapsigargin
achieve steady state
conductances of much longer duration, as in intact monolayers. It was found
that under
3o conditions of basolaterally directed K+ gradients, both forskolin and
thapsigargin activate
macroscopic outwardly rectified (mucosal to serosal) currents at positive
transepithelial
voltages. Experimental I/V relations obtained after forskolin and thapsigargin
stimulation
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displayed reversal potentials {- 40 mV) that approximated the calculated
Nernst-potential (-85
mV calculated as RT/zQo log [Na]o"t~[Na~~n). These results are consistent with
the activation
of distinct cAMP- and Ca++-sensitive basolateral membrane K+ conductances in
conjunction
with one or more nonspecific transepithelial ion shunts, possibly occurring
through
intercellular tight junctions or basolateral membrane "leaks."
To confirm that the observed changes in Isc and G represented K+ transport
through
K+ selective pathways, the effect of forskolin and carbachol on T84 monolayer
conductances
were examined using buffers containing Na+ as the sole permeant cation. These
studies were
performed using an analogous 135 mM basolaterally directed cation (Na+)
gradient. Increases
1 o in Isc and G were not detectable in the absence of K+. Thus, the increases
in cation
conductances induced by agonist stimulation are specific to K+ transport.
Two pharmacologically distinct K+ efflux pathways have been previously
identified in
intact T84 cells. One pathway participates in the secretary response to CAMP-
dependent
agonists and displays sensitivity to Ba++ salts. The other K+ efflux pathway
mediates the
~ 5 response to Ca++-dependent agonists, and is Ba++-insensitive. These
findings were confirmed
in the permeabolized cell model. The CAMP-sensitive IK (elicited by treatment
with forskolin,
pM) was inhibited by greater than 70% by the addition of BaClz (3 mM) to
basolateral
reservoirs. Ba++ , however, had no detectable effect on K+ transport induced
by the
subsequent addition of carbachol ( 100 pM) to the same monolayers. In
contrast, when
2o permeabilized monolayers were treated first with carbachol, the induced
Ca++ IK was inhibited
by 50% by pretreatment with the scorpion venom charybdotoxin (100 nM).
Charybdotoxin,
however, had no detectable effect on K+ transport induced by the subsequent
addition of
forskolin. Thus in permeabilized cells, the differential sensitivity of K+
transport to inhibition
by the K+ channel blockers BaCl2 and charybdotoxin paralleled exactly the
effect of these
25 channel selective inhibitors on K+ transport in intact cells (measured
indirectly as a CY
current).
Taken together, these studies define the permeabilized T84 cell model, and
provide
strong evidence that under the defined conditions both Isc and G represent K+
transport
through distinct cAMP- and Ca++-sensitive basolateral K+ channels.
r a re at le
metabolite, inhibit K+ transport through both cAMP- and Ca++-dependent K+
channels.
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We next tested the hypothesis that clotrimazole may inhibit directly
basolateral
membrane K+ channels in human intestinal T84 cells, as it does in the red
cell. clotrimazole
significantly inhibited the time course of K+ transport after treatment with
the CAMP agonist
forskolin (10 ~,M) and the Ca++ agonist carbachol (100 ~M}. Formal IV
relations taken at
steady state after cAMP or Ca++ stimulation confirm that clotrimazole affected
both cAMP-
and Ca++- sensitive channels. Nearly identical results were obtained with 2-
chlorophenyl-bis-
phenyl methanol. clotrimazole and its metabolite 2-chlorophenyl-bis-phenyl
methanol inhibit
directly both cAMP- and Ca++-sensitive intestinal K+ channels indicating that
the ring
structure in the absence of the imidazole ring sufficient (and perhaps
necessary) for this
1 o bioactivity.
8.4. Clotrimazole targets the basolateral rather than the apical surface of
T84 cells
Methods: Measurement of Cf Conductance of the Apical Plasma Membrane. To
examine apical Cl conductances, CY was used as the sole permeant ion using
identical apical
and basolateral buffer solutions. Monolayers were pemeabilized basolaterally
by the addition
of 100 pM Amphotericin B to the serosal reservoir. Generation of voltage-
current curves of
channel currents were elicited by 1 sec test potentials from -80 to + 80 mV in
10 mV
increments in symmetrical high Choline Cf buffers.
Rest Studies were performed to determine whether the primary target of
2o clotrimazole was located on the basoolateral or apical cell surfaces. Most
rapid inhibition was
achieved by incubation with clotrimazole on both sides of the monolayer.
However,
basolateral application alone was almost as effective as incubation on both
sides.
Additionally, the apparent potency of inhibition of clotrimazole at a fixed
time point was
found to be greater when applied basolaterally than apically. This
preferential action of
clotrimazole at the basolateral surface of the cell is consistent with the
hypothesis that its
principal targets are basolateral K+ channels.
To confirm these findings, we examined Cl- transport in T84 cell monolayers
permeabilized basolaterally with pores formed by amphotericin B. These studies
were
performed with Cl- as the only permeant anion, and with symmetrical apical and
basolateral
3o Cl- concentrations (142 mM). In monolayers not treated with clotrimazole,
the addition of
forskolin (10 ~.M) to basolateral reservoirs increased Cl- conductances
significantly over
baseline, presumably via activation of the cystic fibrosis transmembrane
regulator (CFTR) Cl-
CA 02310773 2000-OS-19
WO 99/26628 PCT/US98/24967
-71 -
channel. In contrast to the clear inhibitory effects of clotrimazole on
basolateral K+
conductances, however, clotrimazole had no detectable effect on either
forskolin- or
thapsigargin-stimulated Cl-conductances. I/V relations for Cl- transport were
nearly identical
in monolayers treated or not treated with clotrimazole. These data provide
further evidence
that clotrimazole inhibits Cl- secretion in intact T84 cell monolayers by
affecting specifically
basolateral K+ channels. Apical membrane Cl-channels are not inhibited.
Ex~a lp a 9: Clotrimazole inhibits Cl- secretion in vivo.
9.1. Chamber studies using rabbit colonic mucosa.
~g hods: 4 male, New Zealand rabbits (2.5 kg) were anesthetized by an
intravenous
injection of pentobarbital (0.5 mllkg). A 15 cm length of distal colon was
removed and
opened longitudinally. External muscle layers were removed by blunt dissection
and colonic
mucosal preparations were mounted in an Ussing chamber (DCTSYS; Precision
Instrument
Design, CA; 10.3 cm'- surface area) and incubated with buffer solution
containing (in mM):
NaCI 122.0, CaCl2, 2.0; MgS04,1.3; KCI, 5.0, glucose, 20; NaHC03, 25.0 (pH
when gassed
with 95% 02/5 CO,; temperature was maintained at 37°C) with and without
clotrimazole
(30~M). The volume of fluid on each side of the mucosa was 7 ml.
Potential difference and Isc were monitored continuously and registered every
10
minutes. Luminal and serosal buffer solutions were interfaced via Ag-AgCI
electrodes
2o (Voltage/Current Clamp, Model VCC600, Physiologic Instruments, Inc., San
Diego, CA,
USA) and Ringer/agar bridge to voltage clamp device (model DVC-1000;
Voltage/Current
Clamp, World Precision Instruments, Inc.). Resistance (R) was calculated using
Ohm's law
and the Isc and is given in ~ x cm2. After stable baseline resistance and Isc
values had been
obtained, mucosal preparations were incubated in the presence or absence of
serosal
clotrimazole (30 pM) for 30 min, and then stimulated by the addition of
forskolin (10 ~M) or
carbachol (10 pM) to the serosal reservoir.
Results: To test the ability of clotrimazole to block K+ channels and thus Cl-
secretion in
native intestinal tissue, we mounted isolated preparations of rabbit colonic
mucosa in Ussing
chambers containing modified Ringer's solution with or without clotrimazole
(30 ~M). After
3o Isc had stabilized, successive additions of forskolin (10 ~.M) and then
cubachol (100 ~,M)
were applied to serosal reservoirs, and Isc and G were monitored continuously.
clotrimazole
inhibited strongly the time course of forskolin induced Isc. Carbachol had no
further effect on
CA 02310773 2000-OS-19
WO 99126628 PCTNS98/24967
-72-
Isc in this system.
9.2. Marine model of secretotonr diarrhea.
Methods: Treated and control, untreated, mice were gavage fed either
clotrimazole
(150 mg/kg/day divided in two equal doses, dissolved in peanut oil at a
concentration of 20
mg/ml) or vehicle control over a 7 day loading period. Mice were then
challenged by gavage
with either 25 ~g purified cholera toxin (Calbiochem, San Diego, CA) in PBS,
vehicle control
alone (PBS without cholera toxin), or cholera toxin in PBS containing 30 ~,M
clotrimazole.
Animals were sacrificed after 5 hours in an uncrowded COZ hood. The carcass
was weighed,
I o the abdomen was opened, and ligatures were tied at the proximal duodenum
and distal rectum.
The intestinal block was dissected free of supporting structures and removed
as a single unit
and weighed. Small and large intestinal segments were normalized to body
weight (intestinal
weight/carcass weight) for each animal.
Results: To examine whether clotrimazole may inhibit intestinal secretion in
vivo, we
utilized a marine model of secretary diarrhea. Balb/C mice were gavage fed 150
mg/kg/day
clotrimazole, divided into two equal doses, or vehicle control every 12 h for
7 days and
subsequently challenged orally with purified cholera toxin (25 fig). Five
hours after treatment
with cholera toxin, the mice were sacrificed and intestinal fluid secretion
assessed
gravimetrically. Pretreatment with clotrimazole reduced by 86% intestinal
fluid secretion
2o induced by cholera toxin. Clotrimazole had no effect on intestinal
secretion in the absence of
cholera toxin. Thus, clotrimazole effectively treated secretory diarrhea in
vivo, presumably
by inhibiting basolateral K+ channels of crypt epithelial cells.
The foregoing written specification is considered to be sufficient to enable
one skilled
in the art to practice the invention. The present invention is not to be
limited in scope by
examples provided, since the examples are intended as a single illustration of
one aspect of
the invention and other functionally equivalent embodiments are within the
scope of the
invention. The advantages and objects of the invention are not necessarily
encompassed by
each embodiment of the invention.
Each of the foregoing patents, patent applications and references is herein
incorporated
3o by reference in its entirety.
What Is Claimed Is:
