Note: Descriptions are shown in the official language in which they were submitted.
CA 02295389 2000-O1-04
APPLICANT: Joy Ann Steele
14334 Park Drive
Edmonton, Alberta, Canada TSR SV2
Citizenship: United States, Canada
TITLE: Method of treatment of nausea, vomiting, and other disorders using
estrogens
BACKGROUND OF THE INVENTION
Field of the Invention
A new use for estrone, derivatives of estrone, equilin and equilenin,
including
pharmaceutically acceptable salts, as well as pharmaceutical compositions
containing these
compounds is claimed.
Description of Related Art
Basic Science Prior Art
The 5-HT3 receptor is a ligand-gated ion channel
The 5-hydroxytryptamine type 3 receptor (5-HT3 receptor) is a member of the
ligand-gated
ion channel superfamily of proteins (Jackson, M.B. & Yakel, J.L., 1995, Ann.
Rev. Physiol.
57:447-468). Ligand-gated ion channels are transmembrane proteins. They appear
to be composed
of five subunits assembled around a central channel that forms a pathway for
ions. Binding of an
agonist leads to the opening of the gate of the ion channel to allow movement
of ions across the
cell membrane. The natural agonist is serotonin (5-hydroxytryptamine, 5-HT).
Two subunits of
the 5-HT3 receptor have been cloned to date (Maricq, A.V., Peterson, A.S.,
Brake, F.J., Myers,
R.M. & Julius, D., 1991, Science, 254:432-437; Davies, P.A., Pistis, M.,
Hanna, M.C., Peters,
CA 02295389 2000-O1-04
J.A., Lambert, J.J., Hales, T.G. & Kirkness, E.F., 1999, Nature 397:359-363).
Several immortal
cell lines express high densities of 5-HT3 receptors including the cell line
that I used in my
experiments (NCB-20). The cDNA cloned from the NCB-20 cell line is nearly
identical (98%
identity) to the cDNA cloned from humans (Maricq et al., 1991 ).
Steroids act on ligand-gated ion channels
The importance of steroids as crucial components of cellular membranes is well
established. Equally well established are the long term endocrine effects of
steroid hormones.
These endocrine effects result from the binding of steroids to intracellular
receptors that
subsequently interact with DNA and modulate gene expression. Less well
delineated, but currently
the subject of increasing interest, are the immediate modulatory effects of
certain steroids on
ligand-gated ion channels (Rupprecht, R. & Holsboer, F., 1999, TINS 22(9):410-
416). For
example, it is now established that steroid anesthetics, as well as certain
endogenous metabolic
products of progesterone and deoxycorticosterone, act on the gamma-
aminobutyric acid type A
receptor (GABAp receptor) at extremely low concentrations. The GABAA receptor
belongs to the
same family of proteins as the 5-HT3 receptor. However, most of the reported
effects of steroids
on ligand-gated ion channels occur at high micromolar concentrations and it is
not clear that these
effects have any physiological relevance or hold any potential for a
therapeutic action. These
nonspecific effects may result from the lipophilic nature of steroids.
Although steroids have long
been used as therapeutic agents for endocrine indications, no steroid is
currently on the market as
an agent that targets a ligand-gated ion channel. Alphaxalone, a steroid
anesthetic that targeted the
GABAp receptor, is the only example and it was withdrawn from the market
because of problems
with the pharmaceutical carrier.
Estrogens are steroids
Estrogens belong to a class of steroids that are responsible for the
development of female
secondary sexual characteristics as well as the differentiation, growth and
functioning of many
tissues in the male. Estrogens also play an important role in the
establishment and maintenance of
pregnancy. The three most abundant estrogens in humans are estradiol, estrone
and estriol. Of the
these three, estradiol is the most potent and is the major secretory product
of the ovary. Estrogens
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are extensively metabolized. For example, estradiol administered orally to
humans is metabolized
to estrone by the gastrointestinal mucosa and the liver (Lievertz, R.W., 1987,
Am. J. Obstet.
Gynecol. 156:1289-1293). During pregnancy, estrogens are synthesized by the
placenta in large
quantities (De Hertogh, R. et al., 1975, J. Clin. Endocrinol. Metab. 40:93-
101).
Estradiol is an antagonist of the 5-HT3 receptor at high concentrations
I have found that estradiol antagonizes the 5-HT3 receptor at high
concentrations. The
concentration of estradiol needed to inhibit the functioning of the 5-HT3
receptor by 50% (ICSO)
was about 3 ~M. This observation was presented in 1994 at the International
Union of
Pharmacologists (IUPHAR) meeting. This observation has also been published by
another group
(Wetzel, C.H.R., et al., 1998, Mol. Endocrinol. 12(9):1441-1451).
Unfortunately, because such
high concentrations of estradiol were needed to cause an effect, the action on
the 5-HT3 receptor is
not of any physiological relevance. Normally, the plasma concentrations of
estrogens are in the
picomolar range. Only during the condition of pregnancy does the plasma level
of estrogens rise
into the low nanomolar range (De Hertogh, et al., 1975). This low affinity
binding site for
estradiol on the 5-HT3 receptor is also not useful as a therapeutic target
since unreasonably high
concentrations of estradiol would have to be administered to a human to exert
an effect on the
5-HTg receptor.
The presence of a low affinity binding site on the 5-HT3 receptor for a
steroid is
predictable from the prior art because many different ligand-gated ion
channels as well as G-
protein coupled receptors have low affinity binding sites for steroids
(Rupprecht & Holsboer,
1999). Since only a few high affinity binding sites have been found, it is not
necessarily
predictable that the 5-HT3 receptor would have a high affinity binding site.
More importantly, it is
certainly not possible to predict the identity of a steroid which would have a
high affinity for a
receptor. This is because the existence of a high affinity binding site must
first be established and
then the structure-activity properties of the binding site must be elucidated.
As detailed later in this
specification, I surprisingly discovered a high affinity binding site on the 5-
HT3 receptor for
estrone. This site is potentially useful as a therapeutic target for the
treatment of nausea and/or
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vomiting and perhaps other, as yet unidentified therapeutic indications. The
reason that this site is
of potential therapeutic value is that only very low amounts of an estrogen
would need to be
administered to a human to acheive a therapeutic effect through an action on
the 5-HT3 receptor.
The 5-HT3 receptor is involved in the control of vomiting in human and in
animals
5-HT3 receptors are widely distributed in the mammalian central, peripheral
and enteric
nervous systems and have also been found on cells of the immune system. The
enteric nervous
system resides within the walls of the gastrointestinal tract. 5-HT3 receptors
have been found to
play an important role in the control of vomiting in a variety of mammals
including humans
(Veyrat-Follet, C., Farinott, R. & Palmer, J.L., 1997, Drugs 53(2):206-234).
The receptors are
present in the part of the brain that is involved in controlling vomiting as
well as in the
gastrointestinal tract. Receptors at both locations have been shown to be
involved in vomiting. It is
thought that 5-HT released from the enterochromaffin cells of the
gastrointestinal mucosa acts on
5-HT3 receptors to initiate the vomiting reflex. Chemotherapy and
radiotherapy, two important
clinical causes of vomiting, may cause release of S-HT from the
enterochromaffm cells.
Chemotherapeutic agents also appear to act directly on the chemoreceptor
trigger zone of the
vomiting center in the brain that then feeds onto neurons containing 5-HT3
receptors to initiate
vomiting.
Currently unidentified physiological roles of 5-HT3 receptors
It is only relatively recently that 5-HT3 receptors were identified. The only
well described
physiological role for these receptors is that of the control of vomiting as
described above.
However, since the receptors are so widely distributed throughout the central
nervous system,
including numerous regions of the brain, it is likely that further research
will identify additional
physiological roles for these receptors. Importantly for the scope of the
invention described in this
specification, it is likely that 5-HT3 receptors may also be found to be
involved in the etiology of
various disorders or diseases. For example, it has been shown that 5-HT3
receptor antagonists
display anxiolytic (Rodgers, R.J., Cole, JC. & Tredwell, J.M., 1995,
Psychopharmacology
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117:306-312) and atypical antipsychotic properties (Zoldan, J., Friedberg, G.,
Goldberg-Stern, H.
& Melamed, E., 1993, Lancet 341:562-563).
Clinical Sciences Background
"Nausea and/or vomiting"
Nausea and vomiting are separate symptoms. Nausea is a feeling of distress and
is often
accompanied by the urge to vomit. Nausea can frequently occur without
vomiting. Vomiting is the
reflex act of disgorging the contents of the stomach through the mouth and is
generally preceded
by nausea. However, vomiting generally occurs with nausea and thus is commonly
referred to as
nausea and vomiting. Vomiting is also called emesis. Pharmaceutical agents
that are employed to
treat vomiting are referred to as antiemetic agents. However, since nausea
usually precedes
emesis, antiemetic agents can, but not necessarily, ameliorate the sensation
of nausea. The phrase
"nausea and/or vomiting" is used throughout the specification and the claims
and is intended to
mean the symptoms of nausea or vomiting occurring together or separately.
Nausea and/or vomiting as a clinical problem
Nausea and/or vomiting are unpleasant and at times limiting side-effects in
several clinical
areas. These symptoms are frequently experienced following the administration
of
chemotherapeutic agents used in the treatment of cancer and other diseases.
The symptoms caused
by the chemotherapeutic agents may be so severe that the patients may refuse
further treatment.
Examples of chemotherapeutic agents for which nausea and/or vomiting are side-
effects include
cisplatin, cyclophosphamide, carboplatin and others. The ability of
chemotherapeutic agent to
induce emesis varies with the agent and ranges from high to low. When used in
combination, the
emetogenic potential of each agent is additive. Cisplatin has the highest
emetogenic potential of
any chemotherapeutic agent. It has also become apparent that chemotherapeutic
agents cause two
distinct phases of nausea and/or vomiting. The first phase occurs shortly
after the administration
of the chemotherapeutic agent and begins to subside by 12 to 24 hours. This
phase is called acute
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nausea and/or vomiting. A second phase of nausea and/or vomiting that develops
24 hours after
chemotherapy is called delayed nausea and/or vomiting. About 1 in 9 patients
experience delayed
nausea and/or vomiting and thus this is a significant clinical problem
(Morrow, G.R., Hickok, J.T.,
Burish, T.G. & Rosenthal, S.N., 1996, Am. J. Clin. Oncol. 19(2):199-203).
The radiotherapy used to treat cancer and other diseases can also cause nausea
and/or vomiting.
The occurence of radiotherapy-induced nausea and/or vomiting is dependent on
several
factors including site, field size and dose per fraction. Clinical
considerations also include prior
surgery and exposure to chemotherapy. Vomiting is more likely to occur when
radiotherapy is
applied to the upper abdomin, when the field size is large and when the dose
per fraction is high.
Post-operative nausea and/or vomiting induced by the use of general
anesthetics is also a
clinical problem. The incidence of symptoms also depends on the site with
abdominal,
gynecological, ear, eye, nose and throat surgeries having the highest
incidence.
Current solutions to the clinical problem of nausea and/or vomiting
There are nine groups of agents that are used clinically for the treatment of
emesis. The
nine groups are: anticholinergics, antihistamines, phenothiazines,
butyrophenones, cannabinoids,
benzamides, glucocorticoids, benzodiazepines and 5-HT3 receptor competitve
antagonists (Merck
Manuel, 1992, Merck Research Laboratories). In general, there are several
agents in each group.
Competitive antagonists of the 5-HT3 receptor are used clinically as
antiemetic agents
The introduction of 5-HT3 receptor competitive antagonists into clinical
practice
revolutionized the treatment of emesis because these agents are more
efficacious and have fewer
side-effects than antiemetic agents from the other groups (Markham, A. &
Sorkin, E.M., 1993,
Drugs, 45:931-952). These agents are: ondansetron (ZofranTM, GlaxoWelcome),
granisetron
(KytrilTM, SmithKline Beecham) and tropisetron (NavobanTM, Sandoz). At the
present time, the
5-HT3 competitive antagonists in combination with the corticosteroid
dexamethasone represent
the best prophylaxis and treatment of acute nausea and/or vomiting.
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Disadvantages of Present Antiemetic Agents
Drugs that act at sites other than the 5-HT3 receptor are not very efficacious
and have poor
side effect profiles. For example, metoclopramide, a benzamide, that acts at
dopamine as well as
5-HT receptors exhibits serious and undesirable side-effects such as extra-
pyramidal effects
characterized by tardive dykinesia, acute dystonias, akathisia and tremor.
Another example is
chlorpromazine, a phenothiazine, which not only has extra-pyramidal side-
effects but cognitive
effects as well. Dexamethasone, a glucocorticoid, is the only steroid used as
an antiemetic agent. It
is not very efficacious and is not generally employed as a single agent. It is
often used in
combination with 5-HT3 receptor competitive antagonists for the prevention of
emesis in the
highly emetogenic therapeutic regimes. Dexamethasone has several unpleasant
side-effects such
as insomnia, dysphoria, fluid retention and muscle weakness. The site of
action for dexamethasone
inducing an antiemetic effect is unknown.
Limitations of the 5-HT3 receptor competitive antagonists as antiemetic agents
Despite the tremendous advance in the management of emesis with the
intoduction of the
5-HT3 receptor competitive antagonists, there is still lots of room for
improvement (Morrow,
G.R., Hickok, J.T. & Roenthal, S.N., 1995, Cancer 76(3):343-357). These agents
have been found
to be ineffective in 40 to 60% of patients. Efficacy appears to be more
pronounced for cisplatin-
containing regimes than for moderate or less emetogenic chemotherapy regimes.
Effectiveness
also appears to be less for delayed nausea and emesis than for acute symptoms.
Control over
nausea appeaxs to be significantly less than control over emesis with control
over nausea
remaining incomplete in approximately half of the patients. As well, the
efficacy of the agents
appears to diminish across repeated days and across repeated chemotherapy
cycles. Interestingly,
the addition of dexamethasone increases the efficacy of both the 5-HT3
receptor competitive
antagonists and other antiemetic agents. Taken altogether, there is still a
need for a safe and
efficacious antiemetic agent.
In addition, 5-HT3 receptor competitive antagonists are extremely expensive.
Consequently, they are not prescribed to the extent that they are needed and
the cost of these
agents is a burden to the health care system.
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Estrone is an old drug and is also an endogenous hormone in both women and
men.
Estrone has been in clinical usage for gynecological (endocrine) indications
for several
decades. Estrone has an excellent side effect profile. No adverse events have
been reported to the
Food and Drug Administration's Adverse Event Reporting System (AERS) as of
October 1999.
Pregnant women have a plasma level of up to 30 nM of unconjugated estrone
during the last six
months of pregnancy (De Hertogh, D., Thomas, K., Bietlot, Y., Vanderheyden, I.
& Ferin, J.,
1975, J. Clin. Endocrinol. Metab. 40:93-101) without any obvious ill effects.
This plasma level is
about three times the level expected to be needed for estrone to exert a
therapeutic effect as an
antiemetic agent as claimed in this invention. Consequently, no adverse side-
effects are
anticipated. Unconjugated estrone is also present in men at a plasma
concentration of 90 to 330
pM.
Estrone as the sole active ingredient has been available as a preparation for
intramuscular
injection (TheelinTM and others) and as a vaginal cream. Estrone is also a
component of the
preparations prescribed for hormone replacement therapy in the treatment of
menopause
(PremarinTM). Premarin also contains several other estrogens: delta-8-estrone,
equilin and
equilenin (Bhavnahi, B.R., 1998, Proc. Soc. Exp. Biol. Med. 217:6-16).
BRIEF SUMMARY OF THE INVENTION
This invention discloses a new use for estrone or derivatives of estrone,
equilin, and
equilenin. The asserted use is an antiemetic agent or for the treatment of
other disorders that can
be ameliorated by noncompetitive antagonism of the 5-hydroxytryptamine type 3
receptor (5-HT3
receptor). Emesis is defined as vomiting and an antiemetic agent is a
therapeutic agent which
prevents or relieves the symptoms of vomiting. Estrone would be administered
to a human or an
animal in need of antiemetic therapy as the active ingredient in a
pharmaceutical composition.
There is still a clinical need for antiemetic agents that are effective and
devoid of undesirable side-
effects. I collected in vitro data that demonstrated that estrone
noncompetitively antagonized the
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functioning of a 5-HT3 receptor at very low nanomolar concentrations. That is,
I discovered a high
affinity steroid binding site on the 5-HT3 receptor. This information had not
been previously
described and cannot be deduced from the prior art. My assertion that estrone
can act as an
antiemetic agent in mammals, including humans, is based on the following
correlations. Since
antagonists of the 5-HT3 receptor are efficacious antiemetic agents in humans
and in animals, and
since estrone is an antagonist of the 5-HT3 receptor, estrone should also
serve as an antiemetic
agent. In addition, estrone appears to be a natural, endogenous antiemetic in
humans being
responsible for the cessation of pregnancy-induced nausea and vomiting.
Further experiments
revealed that the oxygen at carbon 17 of the steroid structure is required for
a high affinity
interaction with the receptor. Consequently, the use of the closely related
estrogens with an
oxygen at carbon 17, equilin and equilenin is also claimed.
DETAILED DESCRIPTION OF THE INVENTION
Estrone has been in clinical usage for decades. Many aspects of its use in
humans such as
safety, side-effects, routes of administration, pharmacokinetics and
biological actions are known.
Estrone is currently not being used as an antiemetic agent. It is in clinical
usage for gynecological
(endocrine) indications only. There are nine groups of agents that are used as
antiemetic agents.
Estrone can be distinguished from eight of these groups by the fact that it
acts on the 5-HT3
receptor while the other eight groups act at other receptors such as dopamine
receptors. The
remaining group of antiemetic agents act at the 5-HT3 receptor by competitive
antagonism of the
agonist binding site. Estrone can be distinguished from these agents since it
acts at the 5-HT3
receptor but by a noncompetitive mechanism.
Advantages of estrone over other groups of antiemetic agents
Competitive antagonists of the 5-HT3 receptor have been found to be the most
efficacious
antiemetic agents. Most of the antiemetic agents in clinical use do not act at
the 5-HT3 receptor.
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Since I have surprising found that estrone is a very potent antagonist of the
5-HT3 receptor, I
anticipate that estrone will be more efficacious than any of the agents that
act at sites other than
the 5-HT3 receptor. Also, estrone has been in clinical usage for decades for
endocrine indications.
It has been found to be safe and virtually free of untoward side-effects. All
of the antiemetic
agents that act at sites other than the 5-HT3 receptor have very unpleasant
side-effects. For
example, metoclopramide induces undesirable problems with movement such as
dystonias.
Antihistamines cause problems with sedation and anticholinergic agents cause
anticholinergic
side-effects. Estrone will not induce problems with dystonias, sedation or
anticholinergic effects.
Advantages of estrone over the 5-HT3 receptor competitive antagonists
Estrone has an advantage over the 5-HT3 receptor competitive antagonists as
its biological
half life is about 4 times longer: 12 to 14 hours compared to 3 to 4 hours
(Compendium of
Pharmaceuticals and Specialties, latest edition; Lievertz, R. W., 1987, Am. J.
Obstet. Gynecol.
156:1289-1293). Also, there are additional routes of adminstration available:
sublingual,
transdermal patch and intramuscular injection. Administration by a transdermal
patch would be
particulary useful in preventing the emesis caused by radiotherapy since these
treatments are
generally given over an extended period of time. Estrone is a natural compound
and is very
inexpensive. The retail price is about 2% that of ondansetron. For a rough
price comparison,
ondansetron sells for about $20 per tablet. Tablets containing estrogens sell
for about $0.20 per
tablet or 1 % of ondansetron tablets. Consequently, the use of estrone as an
antiemetic agent would
result in significant savings to the health care system. The more efficacious
5-HT3 receptor
competitve antagonists are not used as much as they are needed owing to their
cost. Consequently,
a less expensive, efficacious antiemetic agent such as estrone is needed.
Disadvantages to the use of estrone as an antiemetic agent
Estrone would be contraindicated in most estrogen-dependent cancers except
those that
have reached the stage of palliative treatment. Based on the incidence of
various cancers, this
would represent about 10% of the market. Since this is a clearly defined
contraindication, it is not
a very large disadvantage. Estrone would also be strictly contraindicated in
the first trimester of
pregnancy according to the guidelines of the Food and Drug Administration.
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Experimental Data:
Estrone is a noncompetitive antagonist of the 5-HT3 receptor at low nanomolar
concentrations
I surprisingly discovered that a steroid hormone, estrone, antagonises the 5-
HT3 receptor at
extremely low nanomolax concentrations. This action of estrone was previously
unknown and
cannot be predicted from the prior art. This observation demonstrates that a
high affinity binding
site for steroids exists on the 5-HT3 receptor.
I used NCB-20 cells (mouse neuroblastoma x Chinese hamster brain cell line),
an immortal
cell line that expresses 5-HT3 receptors at a high density. Cells were grown
in Dulbecco's
modified Eagles medium containing 10% fetal bovine serum, 100 U/ml penicillin,
100 p,g/ml
streptomycin and 2 mM glutamine. Cells were incubated in 35 mm plates at 37
degrees C in a
humidified 7% carbon dioxide atmosphere. An earlier patch clamp study of NCB-
20 cells showed
that the currents elicited by 5-HT are exclusively carried by 5-HT3 receptors
(Lambent, J.J., Peters,
J.A., Hales, T.G. & Dempster, J., 1989, Br. J. Pharmacol. 97:27-40). I
recorded membrane
currents carried by S-HT3 receptors under voltage-clamp from single cells
using the whole-cell
configuration of the patch clamp technique (Hamill, O.P., Marty, A., Neher,
E., Sakmann, B. &
Sakmann, F.J., 1981, Pfluegers Arch. 391:85-100). Recordings were performed at
a holding
potential of -60 mV. Ligands were applied to the cells using a gravity-feed
rapid perfusion system.
Estrone antagonizes the open conformation of the 5-HT3 receptor at low
nanomolar concentrations
Application of a low concentration of 5-HT (0.5 ~M) elicited an inward current
that did
not decline (desensitize). When the agonist is bound to the receptor and the
receptor is conducting
current, the receptor is in an open conformation. Co-application of estrone
with 5-HT (0.5 p,M)
showed that estrone rapidly and reversibly blocked the current at low
nanomolar concentrations.
That is, estrone antagonized the open conformation of the receptor.
Consequently, the open
conformation has a high affinity binding site for estrone. Concentration-
effect data from 21 cells
were fitted with the Hill (logistic) equation. The concentration of estrone
that inhibited 50% of the
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current (ICso) was found to be 1.5 nM with the standard error of the mean
being 0.2 nM (Steele,
J.A & Martin, LL., 1999, Brit. J. Pharm. 126:Proc. Sup.).
Structure-activity information for the high affinity binding site: a ketone
group at the carbon 17
position is crucial
Comparison of data from three estrogens: estrone, estradiol and estriol gave
some
structure-activity information. Using the co-application protocol described
above, the ICSO for
estradiol was found to be 81 + 12 nM ( 10 cells). Estradiol has a hydroxyl
group at the carbon 17
position on the D ring of the steroid nucleus while estrone has a ketone
group. Since this is the
only difference between the two molecules, it is clear that a ketone group at
the carbon 17 position
is crucial for a high affinity interaction. The ICgo for estriol was found to
be 22 + 8 ~M (8 cells).
Estriol has hydroxyl groups at carbons 16 and 17 and is essentially devoid of
activity. Therefore, a
steroid compound with a ketone group at the carbon 17 position is needed for
activity. Several
closely related compounds to estrone which have a ketone at the carbon 17
position are equilin
and equilenin. These two compounds are estrogens extracted from pregnant
mares' urine and are
contained in the prescription drug Premarin TM (Wyeth-Ayerst). Based on the
data to date, equilin,
equilenin, 2-OH-estrone, 4-OH-estrone, and delta-8-estrone should be active at
the 5-HT3
receptor since they all possess the basic steroid nucleus of an estrogen with
a ketone group at the
carbon 17 position.
Estrone antagonizes the 5-HT3 receptor by a noncompetitive mechanism
The block by estrone was found not to be competitive with the agonist 5-HT.
The ECSo
(concentration of S-HT at which 50% of maximum current amplitude was elicited)
for 5-HT was
2.2 + 0.2 ~M in the absence of estrone (8 cells) and 4.2 + 0.3 pM in the
presence of estrone (8
cells). A noncompetitive antagonist does not shift the ECso for the agonist as
is the case with
estrone. It appears that estrone antagonizes the 5-HT3 receptor through an
allosteric mechanism
and thus differs in its mechanism of block from ondansetron, granisetron, and
tropisetron which
are competitive antagonists.
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The above data that shows that there is a high affinity binding site on the 5-
HT3 receptor
which had not been previously known. A low affinity binding site for estradiol
has been reported
(Wetzel et al., 1998) as described in the Background section. I also found a
low affinity binding
site for estradiol with an ICsp of 3.1 + 0.02 ~M (8 cells) and a low affinity
site for estrone with an
ICsp of 0.3 ~ 0.02 PM (15 cells). These blocking sites were revealed by
applying the estrogens
before the 5-HT ( 10 p,M) was applied. The estrogens were then interacting
with the agonist-free
non-conducting state of the receptor. This is also called the closed
conformation of the receptor.
Consequently, it is only when the receptor is in the agonist-bound, conducting
state that the high
affinity binding site is available. Conformation-dependent binding site
affinities for ion channels
are well known (Hille, B., 1992, Ionic Channels of Excitable Membranes,
Sinauer Assoc. Inc., pp.
390-422). However, it could not be predicted that such a high affinity binding
site would be
present on the S-HT3 receptor. This was an unexpected discovery. It is
important to point out that
it is the high affinity binding site that enables this disclosed invention to
be useful. It is this site
that can be a target for a therapeutic agent. This is because only a small
amount of an estrogen
would have to be administered to achieve a high enough concentration in the
plasma to be
therapeutically effective.
Suggested biological activity based on the in vitro experimental data: Estrone
exhibits antiemetic
activity
The asserted utility of estrone or derivatives of estrone as well as equilin
and equilenin as
an antiemetic agents in mammals including humans is based on two correlations.
First, antagonists
of the 5-HT3 receptor such as ondansetron are in clinical usage as antiemetic
agents. Therefore,
since estrone also antagonizes the S-HT3 receptor, it should also act as an
antiemetic agent.
Second, estrone appears to be an endogenous antiemetic in humans. I suggest
that estrone plays an
important physiological role in the cessation of pregnancy-induced emesis
('morning sickness').
The plasma levels of free, unconjugated estrone are low during the first
trimester of pregnancy
when at least 50% of women experience nausea and vomiting (Klebanoff, M.A.,
Koslowe, P.A.,
Kaslow, R. & Rhoads, G.G., 1985, Obstet. Gynecol. 66:612-616). The plasma
level of estrone
rises dramatically at the end of the first trimester as a result of increased
synthesis by the placenta
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(De Hertogh et al., 1975). This dramatic rise in the levels of estrone
corresponds to the time of the
cessation of morning sickness. The mean concentration of estrone at this time
during pregnancy
(12 weeks) was found to be 3.7 nM which is about the same as the ICsp (1.5 nM)
that I measured
in the in vitro preparation. This extremely close correlation suggests that
estrone is responsible for
resolving the nausea and/or vomiting induced by pregnancy.
Contemplated Modes of Use
My discovery of a high affinity binding site for estrogens on the 5-HT3
receptor which is
distinct from the agonist site, is the novel aspect of this invention.
Consequently, any ligand that is
capable of noncompetitively antagonizing the 5-HT3 receptor with an ICsp value
less than 75 nM
is meant to be included within the scope of this invention. Based on the data
collected to date, a
compound selected from a group comprising estrone, 2-OH-estrone, 4-OH-estrone,
delta-8-
estrone, an obvious chemical derivative of estrone, equilin and equilenin
could be used as a
therapeutic agent in humans or in animals. It is intended to be understood
that the compound must
be formulated into a pharmaceutical composition appropriate to the route of
administration for
administration to a human or an animal. Currently, the only well described
therapeutic use for an
antagonist of the 5-HT3 receptor is that of an antiemetic agent. However, it
is likely that additional
therapeutic uses for 5-HT3 antagonists will be identified. Consequently, any
disease or disorder
that may be treated with a 5-HT3 receptor noncompetitive antagonist is meant
to be included
within the scope of the present invention.
Currently identified therapeutic indications for a 5-HT3 antagonist
5-HT3 receptor antagonists are currently used as antiemetic agents. Thus, a
method of
treatment to prevent or to provide relief from nausea andlor vomiting by
administering a 5-HT3
receptor noncompetitive antagonist to a human or an animal is claimed. Nausea
and/or vomiting
occurs as a clinical problem as a result of administration of chemotherapeutic
agents to treat
cancer or other diseases. It also occurs as a result of radiotherapy to treat
cancer or other diseases.
In addition, general anesthetic agents used during surgery cause nausea and/or
vomiting.
14
CA 02295389 2000-O1-04
Currently unidentified therapeutic indications for a 5-HT3 receptor
noncompetitive antagonist
The physiological roles of the S-HT3 receptors have not been exhaustively
described. It is
likely that further research will uncover additional physiological roles for
this receptor.
Consequently, it is also likely that 5-HT3 receptors may be found to involved
in the etiology of
disorders or diseases other than those that involve vomiting. Since 5-HT3
receptors are located in
the central, peripheral and enteric nervous systems, and cells of the immune
system (Khan, N.A.
& Poisson, J.P., 1999, J. Neuroimmunol. 99(1):53-60), it is likely that the
disorders or diseases
will involve these systems specifically. It is also possible that the symptoms
of these disorders or
diseases may be ameliorated by noncompetitively antagonizing the functioning
of 5-HT3
receptors. For example, it has been suggested that 5-HT3 receptor antagonists
may be useful in
treating various behavioral disorders (Apud, J.A., 1993,
Neuropsychopharmacology 8:117-130).
These antagonists have shown some anxiolytic (Rodgers, R.J., Cole, J.C. &
Tredwell, J.M., 1995,
Psychopharmacology 117:306-312) and atypical antipsychotic properties (Zoldan,
J., Friedberg,
G., Goldberg-Stern, H. & Melamed, E., 1993, Lancet 341:562-563). It is
intended that these
currently unidentified therapeutic indications be included within the scope of
the claims of this
invention.
The term "disorder" means an abnormal physical or mental condition that
includes but is
not limited to disorders of gastrointestinal motility, as well as mood,
thought, and behavioral
disorders. The symptoms include but are not limited to diarrhea and related
symptoms such as
decreased absorption times, depressed moods, cognitive problems such as mental
slowing,
problems with attention such as inability to concentrate and headache.
Active ingredient
The term active ingredient means one of the named compounds or its
pharmaceutically
acceptable salt. Estrone, delta-8-estrone, 2-OH-estrone, 4-OH-estrone, equilin
and equilenin are
commercially available. Pharmaceutically acceptable salts of these compounds
can be formed
according to conventional means known in the art. Estrone, equilin, equilenin,
and delta-8-estrone
CA 02295389 2000-O1-04
are contained in the currently available prescription drug Premarin TM
(Bhavnani, B., 1998, Proc.
Soc. Exp. Biol & Med. 217:6-16).
The systematic names of the named compounds are:
Estrone is 3-hydroxyestra-1,3,5(10)-trim-17-one.
Estradiol is estra-1,3,5(10)-triene-3,17B-diol.
Delta-8-estrone is 3-hydroxyestra-1,3,5(10),8-tetraen-17-one.
4-OH estrone is 4-hydroxyestra-1,3,5(10)-trim-17-one.
2-OH estrone is 2-hydroxyestra-1,3,5(10)-trim-17-one.
Equilin is 3-hydroxyestra-1,3,5(10),7-tetraen-17-one.
Equilenin is 3-hydroxyestra-1,3,5,7,9-pentaen-17-one.
A "chemical derivative" of estrone according to the invention is a compound
which
exhibits a chemical difference but is a noncompetitive antagonist of the 5-HT3
receptor with an
ICsp value less than 75 nM. Such a compound must also demonstrate a tolerable
side-effect profile
when administered to a human or an animal at a dose sufficient to provide
relief from symptoms.
Some chemical derivatives of estrone are 4-OH estrone, 2-OH estrone, and delta-
8-estrone. Other
chemical derivatives of estrone can be prepared using standard chemical
methodology. For
example, halogenation of the rings of the steroid structure have been found to
provide useful
therapeutic agents. The rings can be halogenated at various positions with
chlorine, fluorine,
iodine or bromine atoms. For example, clomestrone (16-chloro-3-methoxyestra-
1,3,5-(10)-trien-
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CA 02295389 2000-O1-04
17-one) is chlorinated at the carbon 16 position. This compound was previously
used as a
therapeutic agent.
A "pharmaceutically acceptable salt" of the active ingredient according to the
invention is
understood to mean both inorganic and organic salts, such as described in
Remington's
Pharmaceutical Sciences (latest edition). Although the free-base forms of the
compounds can be
used in the methods of the present invention, it may be preferred to prepare
and use a
pharmaceutically acceptable salt form. The compounds named in this invention
form
pharmaceutically acceptable acid and base addition salts with a wide variety
of inorganic, and
preferably, organic salts and include the physiologically acceptable salts
that are often used in
pharmaceutical chemistry. Such salts are also part of this invention. Typical
inorganic salts used to
form such salts include hydrochloric, hydrobromic, nitric, sulfuric and the
like. Salts derived from
organic acids may also be used. Such pharmaceutically acceptable salts include
but are not limited
to acetate, benzoate, citrate, cypionate, enanthate, undecylate, valerate,
maleate, proprionate,
deconate, sulfate, sulfonate, fumarate, glucoronate and salts with
physiologically tolerable amines.
These salts can be prepared as esters of the estrogens according to
conventional methods known to
a person skilled in the art. The salts of the esters of estrogens are often
referred to as conjugated
estrogens. Conjugated estrogens may be derived wholly or in part from equine
urine or may be
prepared synthetically. A pharmaceutically acceptable salt of estrone
additionally includes
estropipate which is also referred to as piperazine estrone sulfate.
Estropipate is contained in the
pharmaceutical composition OgenTM (Pharmacia & Upjohn) which is prescribed for
hormone
replacement therapy in the treatment of menopausal symptoms. Consequently, the
active
ingredient according to the invention may be a compound other than estrone.
A "metabolic precursor" of estrone is defined as any compound administered to
a human
or an animal that is metabolized to estrone. For example, it is known that
estradiol when orally
administered to a human is metabolized to estrone by the gastrointestinal
mucosa and the liver
(Lievertz, R.W., 1987, Am. J. Obstet. Gynecol. 156:1289-1293).
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CA 02295389 2000-O1-04
It is meant to be understood that any of the possible stereoisomers of the
compound that is
selected as the active ingredient is included within the scope of the
invention. A stereoisomer is a
compound in which the atoms are linked in the same order but differ in their
spatial arrangements.
For example, estrone has chiral centers and therefore can exist in numerous
stereoisomeric forms.
For purposes of this invention, the name of the compound is meant to include
all stereoisomeric
forms of the compound. The compound could be used in a pure form or as a
mixture of
stereoisomers. Stereoisomers can generally be prepared following procedures
known in the art.
Pharmaceutical compositions containing the active ingredient
The term "pharmaceutical composition" means combining the active ingredient or
a
pharmaceutically acceptable salt with one or more pharmaceutically acceptable
carriers, diluents
or excipients to form a pharmaceutical formulation or preparation suitable for
administration to a
human or an animal. A pharmaceutical composition can be prepared by procedures
known in the
art using well known and readily available ingredients. The pharmaceutical
composition will vary
according to the route of administration, e.g. intramuscular, intravenous,
oral, transdermal,
sublingual, etc. Standard methods of formulating compounds for administration
as
pharmaceuticals can be found in Remington's Pharmaceutical Sciences, Mack
Publishing
Company, Easton, PA, latest edition.
The term "pharmaceutically acceptable" means that the carriers, diluents,
excipients and/or
salts must be compatible with the other ingredients of the composition, and
not deleterious to the
recipient.
Non-parenteral administration
Oral
For oral administration, the active ingredient is generally administered as a
tablet, capsule,
or syrup and pharmaceutically acceptable nontoxic compositions are formed
using the normally
employed excipients auch as pharmaceutical grades of mannitol, lactose,
starch, magnesium
stearate, talcum, cellulose, magnesium carbonate, and so forth. The active
ingredient may also be
micronized in a fluid energy mill to decrease the particle size. It has been
found that preparing
18
CA 02295389 2000-O1-04
estradiol in a micronized form increases its absorption from the
gastrointestinal tract and this is
likely to hold true for the compounds named in the methods of the present
invention.
Sublingual
For sublingual administration, the active ingredient is generally administered
as a tablet
and pharmaceutically acceptable compositions are formed as detailed above for
oral
administration.
Suppositories
For administration via suppository, conventional binders and carriers include,
for example,
polyalkylene glycols or triglycerides, and the suppositories generally contain
active ingredient in
the range of 0.5 to 10%.
Parenteral
Parenteral administration refers to any route that does not involve the
intestinal tract and is
characterized by injection, whether subcutaneously, intramuscularly, or
intravenously, by
transdermal delivery via a patch, or by implantation of subcutaneous pellets.
Injection
Injectables can be prepared in conventional forms, either as solutions or
suspensions, in
solid forms suitable for solution or suspension in liquid prior to injections
or as emulsions.
Suitable excipients include water, saline, dextrose, glycerol, and the like.
If desired, the
pharmaceutical compositions may also include minor amounts of nontoxic
auxiliary substances,
such as wetting agents or emulsifying agents, pH-buffering agents and so
forth. Estrone has been
available as a prescription drug over the past several decades in a
pharmaceutical composition for
intramuscular injection, for example, TheelinTM (Parkedale) and many others.
Transdermal delivery
A transdermal patch may be used to deliver the active ingredient in accordance
with
known techniques. The patch is typically applied for a long period, e.g. 1 to
4 days, allowing a
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CA 02295389 2000-O1-04
slow and constant delivery of the active ingredient. This may be a preferred
route of
administration when nausea and vomiting is induced by radiotherapy.
Radiotherapy for the
treatment of cancer or other diseases is generally given over the course of
days to weeks and the
constant supply of an antiemetic agent by a transdermal patch would be a
convenient method of
treatment. In a transdermal patch, the patch itself maintains the active
ingredient in contact with
the desired localized skin surface. A solvent system for a transdermal patch
may include, for
example, oleic acid, linear alcohol lactate and dipropylene glycol, or other
solvent systems known
in the art. The active ingredient may be dissolved or suspended in the
carrier.
Determination of dose
The term "effective amount" is defined as the dose that prevents or provides
substantial
relief from nausea and/or vomiting or relieves the symptoms of other diseases
or disorders.
A proposed dose of the active ingredient for administration in man (of
approximately 70
kg body weight) is 0.05 to SO mg, preferably 0.1 to 15 mg of active ingredient
per unit dose which
could be administered, for example, 1 to 4 times per day. The dose could be
predetermined by
testing in the human model of emesis which uses syrup of Ipecac as the emetic
agent (Minton, N.
et al., 1993, Clin. Pharmacol. Ther. 54:53-57). This model has shown good
correlation between
effective dose to protect against ipecac-induced vomiting and the effective
dose to protect against
vomiting induced by chemotherapeutic agents. The dose will depend on the route
of
administration and the body weight of the patient. It will be appreciated that
it may be necessary to
make routine variations to the dosage depending on the age and the weight of
the patient as well as
the severity of the condition to be treated.
The pharmaceutical composition containing the active ingredient according to
this
invention may be used prophylactically and references in this specification to
treatment include
prophylactic treatment as well as alleviation of acute symtoms.
The pharmaceutical composition containing the active ingredient according to
this
invention may be used in combination with other antiemetic agents such as but
not limited to
CA 02295389 2000-O1-04
ondansetron (ZofranTM, Glaxo-Welcome), granisetron (KytrilTM, Smith-Kline
Beecham) or
tropisetron (NavobanTM, Sandoz).
It should be understood that various changes and modifications to the
presently preferred
embodiments described herein will be apparent to those skilled in the art.
Such changes and
modifications can be made without departing from the spirit and the scope of
the present invention
and without diminishing its attendant advantages. It is therefore intended
that such changes and
modifications be covered by the appended claims.
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