Title: Methods for treating
gastroesophageal reflux disease
United States Patent: 7,361,646
Issued: April 22, 2008
Inventors: Belanoff; Joseph
K. (Woodside, CA)
Therapeutics, Inc. (Menlo Park, CA)
Filed: November 5, 2003
This invention relates to the discovery
that agents capable of inhibiting the biological action of the
glucocorticoid receptor can be used in methods for treating
gastroesophageal reflux disease in a subject.
Description of the
BRIEF SUMMARY OF THE INVENTION
The present invention is based at least in part, upon the discovery that
administration of a glucocorticoid receptor antagonist provides an effective
and improved treatment for gastroesophageal reflux disease. Thus, in one
aspect, the invention is directed toward methods of treating
gastroesophageal reflux disease in a subject, provided that the subject is
not otherwise in need of treatment with a glucocorticoid receptor
In one aspect of the invention, the glucocorticoid receptor antagonist
comprises a steroidal skeleton with at least one phenyl-containing moiety in
the 11-beta position of the steroidal skeleton. In one aspect, the
phenyl-containing moiety in the 11-beta position of the steroidal skeleton
is a dimethylaminophenyl moiety. In another aspect, the glucocorticoid
receptor antagonist is mifepristone.
In one aspect of the present invention, the glucocorticoid receptor
antagonist is selected from the group consisting of
In another aspect, the glucocorticoid receptor antagonist is selected from
the group consisting
In another one aspect, the glucocorticoid receptor antagonist is
In another aspect of the present invention, the glucocorticoid receptor
antagonist is administered in a daily amount of between about 0.5 to about
35 mg per kilogram of body weight per day. In another aspect, the
glucocorticoid receptor antagonist is administered in a daily amount of
between about 5 to about 15 mg per kilogram of body weight per day.
In one aspect of the present invention, the administration is once per day.
In yet another aspect, the mode of administration is by a transdermal
application, by a nebulized suspension, or by an aerosol spray. In another
aspect, the mode of administration is oral.
In another aspect the invention also provides a kit for treating
gastroesophageal reflux disease in a subject. The kit comprises a specific
glucocorticoid receptor antagonist and an instructional material teaching
the indications, dosage and schedule of administration of the glucocorticoid
receptor antagonist to a patient suffering from gastroesophageal reflux
DETAILED DESCRIPTION OF THE INVENTION
This invention pertains to the surprising discovery that agents that can
inhibit glucocorticoid-induced biological responses are effective for
treating gastroesophageal reflux disease. In treating gastroesophageal
reflux disease, the methods of the invention can ameliorate, eliminate,
reduce or prevent the symptoms of gastroesophageal reflux disease. In one
embodiment, the methods of the invention use agents that act as GR
antagonists, blocking the interaction of cortisol with GR, to treat
gastroesophageal reflux disease. The methods of the invention are effective
in treating gastroesophageal reflux disease in an afflicted patient.
Cortisol acts by binding to an intracellular, glucocorticoid receptor (GR).
In humans, glucocorticoid receptors are present in two forms: a ligand-binding
GR-alpha of 777 amino acids; and, a GR-beta isoform that differs in only the
last fifteen amino acids. The two types of GR have high affinity for their
specific ligands, and are considered to function through the same signal
The biological effects of cortisol, including pathologies or dysfunctions
caused by hypercortisolemia, can be modulated and controlled at the GR level
using receptor antagonists. Several different classes of agents are able to
act as GR antagonists, i.e., to block the physiologic effects of GR-agonist
binding (the natural agonist is cortisol). These antagonists include
compositions, which, by binding to GR, block the ability of an agonist to
effectively bind to and/or activate the GR. One family of known GR
antagonists, mifepristone and related compounds, are effective and potent
anti-glucocorticoid agents in humans (Bertagna, J. Clin. Endocrinol. Metab.
59:25, 1984). Mifepristone binds to the GR with high affinity, with a K of
dissociation <10.sup.-9 M (Cadepond, Annu. Rev. Med. 48:129, 1997). Thus, in
one embodiment of the invention, mifepristone and related compounds are used
to treat gastroesophageal reflux disease in a subject.
As the methods of the invention include use of any means to inhibit the
biological effects of an agonist-bound GR, illustrative compounds and
compositions which can be used to treat gastroesophageal reflux disease in a
subject are also set forth. Routine procedures that can be used to identify
further compounds and compositions able to block the biological response
caused by a GR-agonist interaction for use in practicing the methods of the
invention are also described. As the invention provides for administering
these compounds and compositions as pharmaceuticals, routine means to
determine GR antagonist drug regimens and formulations to practice the
methods of the invention are set forth below.
II. Diagnosis of Gastroesophageal Reflux Disease in a Subject
Gastroesophageal reflux disease (GERD) is characterized by heartburn and
regurgitation, which may also include dysphagia. The heartburn
characteristic of GERD is most frequently described as a sub-sternal burning
that occurs after meals and often worsens when lying down. Other symptoms
that may be associated with GERD include, but are not limited to atypical
chest pain, hoarseness, nausea, cough, odynophagia and asthma.
Diagnosis may be made from the presentation of the characteristic GERD
symptoms alone, but sometimes further tests are needed to confirm the
diagnosis of GERD. In cases wher further diagnosis is warranted, the further
diagnosis is typically made by treating patients with medications that
suppress the production of acid by the stomach. Acid suppressing medications
include proton pump inhibitors such as Prilosec (omeprazole), Prevacid (lansoprazole),
Aciphex (rabeprazole), Protonix (pantoprazole), and Nexium (esomeprazole),
and histamine blockers such as Zantac (ranitidine), Tagamet (cimetidine),
and Pepcid (famotidine). If the heartburn then is diminished to a large
extent, a diagnosis of GERD may be confirmed.
In some cases further diagnostic measures may be carried out. For example,
if doubts remain about the diagnosis the after the above tests are
completed, or if complications are a concern. The gold standard for
diagnosing GERD is esophageal acid testing.
Patients with the symptoms or complications of GERD have reflux of more
acid, and the acid remains longer in the esophagus when compared to healthy
individuals. Thus, diagnosis of GERD can be confirmed or extended by
performing a 24-hour esophageal pH test. A pH monitor is placed in the
esophagus above the lower esophageal sphincter, and the pH is recorded at
regular intervals over a 24-hour test period. Combined with a diary of
symptoms kept by the patient, this method permits GERD to be diagnosed and
correlated with the lowering of esophageal pH that occurs with reflux.
A method for prolonged measurement (48 hours) of acid exposure in the
esophagus may also be conducted. The method utilizes a small, wireless
capsule that is attached to the esophagus just above the LES. The capsule
measures the acid refluxing into the esophagus and transmits this
information to a receiver that is worn at the waist. At the completion of
the test, the information from the receiver is downloaded into a computer
and analyzed. The capsule falls off of the esophagus after 3-5 days and is
passed in the stool.
III. General Laboratory Procedures
When practicing the methods of the invention, a number of general laboratory
tests can be used to assist in the diagnosis, progress and prognosis of the
patient with gastroesophageal reflux disease, including monitoring of
parameters such as blood cortisol, drug metabolism, brain structure and
function and the like. These procedures can be helpful because all patients
metabolize and react to drugs uniquely. In addition, such monitoring may be
important because each GR antagonist has different pharmacokinetics.
Different patients and disease conditions may require different dosage
regimens and formulations. Such procedures and means to determine dosage
regimens and formulations are well described in the scientific and patent
literature. A few illustrative examples are set forth below.
a. Determining Blood Cortisol Levels
The invention may be practiced upon patients with apparently normal levels
of blood cortisol. However, since the treatment for gastroesophageal reflux
disease comprises administration of a glucocorticoid receptor antagonist,
monitoring blood cortisol and determining baseline cortisol levels are
useful laboratory tests to aid in the diagnosis, treatment and prognosis of
a gastroesophageal reflux disease patient. A wide variety of laboratory
tests exist that can be used to determine whether an individual is normal,
hypo- or hypercortisolemic. Patients with gastroesophageal reflux disease
typically have normal levels of cortisol that are often less than 25 .mu.g/dl
in the morning, and frequently about 15 .mu.g/dl or less in the afternoon,
although the values often fall at the high end of the normal range, which is
generally considered to be 5-15 .mu.g/dl in the afternoon.
Immunoassays such as radioimmunoassays are commonly used because they are
accurate, easy to do and relatively cheap. Because levels of circulating
cortisol are an indicator of adrenocortical function, a variety of
stimulation and suppression tests, such as ACTH Stimulation, ACTH Reserve,
or dexamethasone suppression (see, e.g., Greenwald, Am. J. Psychiatry
143:442-446, 1986), can also provide diagnostic, prognostic or other
information to be used adjunctively in the methods of the invention.
One such assay available in kit form is the radioimmunoassay available as
"Double Antibody Cortisol Kit" (Diagnostic Products Corporation, Los
Angeles, Calif.), (Acta Psychiatr. Scand. 70:239-247, 1984). This test is a
competitive radioimmunoassay in which .sup.125I-labeled cortisol competes
with cortisol from an clinical sample for antibody sites. In this test, due
to the specificity of the antibody and lack of any significant protein
effect, serum and plasma samples require neither preextraction nor
predilution. This assay is described in further detail in Example 2, below.
b. Determination of Blood/Urine Mifepristone Levels
Because a patient's metabolism, clearance rate, toxicity levels, etc.
differs with variations in underlying primary or secondary disease
conditions, drug history, age, general medical condition and the like, it
may be necessary to measure blood and urine levels of GR antagonist. Means
for such monitoring are well described in the scientific and patent
literature. As in one embodiment of the invention mifepristone is
administered to treat gastroesophageal reflux disease, an illustrative
example of determining blood and urine mifepristone levels is set forth in
the Example below.
c. Other Laboratory Procedures
Laboratory tests monitoring and measuring GR antagonist metabolite
generation, plasma concentrations and clearance rates, including urine
concentration of antagonist and metabolites, may also be useful in
practicing the methods of the invention. For example, mifepristone has two
hydrophilic, N-monomethylated and N-dimethylated, metabolites. Plasma and
urine concentrations of these metabolites (in addition to RU486) can be
determined using, for example, thin layer chromatography, as described in
Kawai Pharmacol. and Experimental Therapeutics 241:401-406, 1987.
IV. Glucocorticoid Receptor Antagonists to Treat Gastroesophageal Reflux
Disease in a Subject
The invention provides for methods for treating gastroesophageal reflux
disease in a subject utilizing any composition or compound that can block a
biological response associated with the binding of cortisol or a cortisol
analogue to a GR. Antagonists of GR activity utilized in the methods of the
invention are well described in the scientific and patent literature. A few
illustrative examples are set forth below.
A. Steroidal Anti-Glucocorticoids as GR Antagonists.
Steroidal glucocorticoid antagonists are administered to treat
gastroesophageal reflux disease in various embodiments of the invention.
Steroidal antiglucocorticoids can be obtained by modification of the basic
structure of glucocorticoid agonists, i.e., varied forms of the steroid
backbone. The structure of cortisol can be modified in a variety of ways.
The two most commonly known classes of structural modifications of the
cortisol steroid backbone to create glucocorticoid antagonists include
modifications of the 11-beta hydroxy group and modification of the 17-beta
side chain (see, e.g., Lefebvre, J. Steroid Biochem. 33:557-563, 1989).
Examples of steroidal GR antagonists include androgen-type steroid compounds
as described in U.S. Pat. No. 5,929,058, and the compounds disclosed in U.S.
Pat. Nos. 4,296,206; 4,386,085; 4,447,424; 4,477,445; 4,519,946; 4,540,686;
4,547,493; 4,634,695; 4,634,696; 4,753,932; 4,774,236; 4,808,710; 4,814,327;
4,829,060; 4,861,763; 4,912,097; 4,921,638; 4,943,566; 4,954,490; 4,978,657;
5,006,518; 5,043,332; 5,064,822; 5,073,548; 5,089,488; 5,089,635; 5,093,507;
5,095,010; 5,095,129; 5,132,299; 5,166,146; 5,166,199; 5,173,405; 5,276,023;
5,380,839; 5,348,729; 5,426,102; 5,439,913; 5,616,458, 5,696,127, and
6,303,591. Such steroidal GR antagonists include cortexolone,
dexamethasone-oxetanone, 19-nordeoxycorticosterone, 19-norprogesterone,
y-4,9-estradien-3-one (RU009), and
Other examples of steroidal antiglucocorticoids are disclosed in Van Kampen
et al. (2002) Eur. J. Pharmacol. 457(2-3):207, WO 03/043640, EP 0 683 172
B1, and EP 0 763 541 B1, each of which is incorporated herein by reference.
EP 0 763 541 B1 and Hoyberg et al., Int'l J. of Neuro-psychopharmacology,
5:Supp. 1, S148 (2002); disclose the compound
stra-4,9-dien-3-one (ORG 34517) which in one embodiment, is administered in
an amount effective to treat gastroesophageal reflux disease in a subject.
1. Removal or Substitution of the 11-beta Hydroxy Group
Glucocorticoid antagonists with modified steroidal backbones comprising
removal or substitution of the 11-beta hydroxy group are administered in one
embodiment of the invention. This class includes natural antiglucocorticoids,
including cortexolone, progesterone and testosterone derivatives, and
synthetic compositions, such as mifepristone (Lefebvre, et al. supra).
Preferred embodiments of the invention include all 11-beta-aryl steroid
backbone derivatives because these compounds are devoid of progesterone
receptor (PR) binding activity (Agarwal, FEBS 217:221-226, 1987). Another
preferred embodiment comprises an 11-beta phenyl-aminodimethyl steroid
backbone derivative, i.e., mifepristone, which is both an effective anti-glucocorticoid
and anti-progesterone agent. These compositions act as reversibly-binding
steroid receptor antagonists. For example, when bound to a 11-beta phenyl-aminodimethyl
steroid, the steroid receptor is maintained in a conformation that cannot
bind its natural ligand, such as cortisol in the case of GR (Cadepond, 1997,
Synthetic 11-beta phenyl-aminodimethyl steroids include mifepristone, also
known as RU486, or
-4,9-dien-3-one). Mifepristone has been shown to be a powerful antagonist of
both the progesterone and glucocorticoid (GR) receptors. Another 11-beta
phenyl-aminodimethyl steroids shown to have GR antagonist effects includes
-4,9-estradien-3-one) (see Bocquel, J. Steroid Biochem. Molec. Biol.
45:205-215, 1993). Another GR antagonist related to RU486 is RU044
1993, supra). See also Teutsch, Steroids 38:651-665, 1981; U.S. Pat. Nos.
4,386,085 and 4,912,097.
One embodiment includes compositions containing the basic glucocorticoid
steroid structure which are irreversible anti-glucocorticoids. Such
compounds include alpha-keto-methanesulfonate derivatives of cortisol,
including cortisol-21-mesylate (4-pregnene-11-beta, 17-alpha, 21-triol-3,
20-dione-21-methane-sulfonate and dexamethasone-21-mesylate
(16-methyl-9alpha-fluoro-1,4-pregnadiene-11 beta, 17-alpha, 21-triol-3,
20-dione-21-methane-sulfonte). See Simons, J. Steroid Biochem. 24:25-32,
1986; Mercier, J. Steroid Biochem. 25:11-20, 1986; U.S. Pat. No. 4,296,206.
2. Modification of the 17-beta Side Chain Group
Steroidal antiglucocorticoids which can be obtained by various structural
modifications of the 17-beta side chain are also used in the methods of the
invention. This class includes synthetic antiglucocorticoids such as
dexamethasone-oxetanone, various 17, 21-acetonide derivatives and
17-beta-carboxamide derivatives of dexamethasone (Lefebvre, 1989, supra;
Rousseau, Nature 279:158-160, 1979).
3. Other Steroid Backbone Modifications
GR antagonists used in the various embodiments of the invention include any
steroid backbone modification which effects a biological response resulting
from a GR-agonist interaction. Steroid backbone antagonists can be any
natural or synthetic variation of cortisol, such as adrenal steroids missing
the C-19 methyl group, such as 19-nordeoxycorticosterone and
19-norprogesterone (Wynne, Endocrinology 107:1278-1280, 1980).
In general, the 11-beta side chain substituent, and particularly the size of
that substituent, can play a key role in determining the extent of a
steroid's antiglucocorticoid activity. Substitutions in the A ring of the
steroid backbone can also be important. 17-hydroxypropenyl side chains
generally decrease antiglucocorticoid activity in comparison to 17-propinyl
side chain containing compounds.
Additional glucocorticoid receptor antagonists known in the art and suitable
for practice of the invention include 21-hydroxy-6,19-oxidoprogesterone (see
Vicent, Mol. Pharm. 52:749-753, 1997), Org31710 (see Mizutani, J Steroid
Biochem Mol Biol 42(7):695-704, 1992), RU43044, RU40555 (see Kim, J Steroid
Biochem Mol Biol. 67(3):213-22, 1998), RU28362, and ZK98299.
B. Non-Steroidal Anti-Glucocorticoids as Antagonists.
Non-steroidal glucocorticoid antagonists are also used in the methods of the
invention to gastroesophageal reflux disease in a subject. These include
synthetic mimetics and analogs of proteins, including partially peptidic,
pseudopeptidic and non-peptidic molecular entities. For example, oligomeric
peptidomimetics useful in the invention include (alpha-beta-unsaturated)
peptidosulfonamides, N-substituted glycine derivatives, oligo carbamates,
oligo urea peptidomimetics, hydrazinopeptides, oligosulfones and the like
(see, e.g., Amour, Int. J. Pept. Protein Res. 43:297-304, 1994; de Bont,
Bioorganic & Medicinal Chem. 4:667-672, 1996). The creation and simultaneous
screening of large libraries of synthetic molecules can be carried out using
well-known techniques in combinatorial chemistry, for example, see van
Breemen, Anal Chem 69:2159-2164, 1997; and Lam, Anticancer Drug Des
12:145-167, 1997. Design of peptidomimetics specific for GR can be designed
using computer programs in conjunction with combinatorial chemistry
(combinatorial library) screening approaches (Murray, J. of Computer-Aided
Molec. Design 9:381-395, 1995; Bohm, J. of Computer-Aided Molec. Design
10:265-272, 1996). Such "rational drug design" can help develop peptide
isomerics and conformers including cycloisomers, retro-inverso isomers,
retro isomers and the like (as discussed in Chorev, TibTech 13:438-445,
Examples of non-steroidal GR antagonists include ketoconazole, clotrimazole;
N-(2-chlorotrityl)-L-prolinol acetate; 1-(2-chlorotrityl)-2-methylimidazole;
N-((2,6-dichloro-3-methylphenyl)diphenyl)methylimidazole (see U.S. Pat. No.
6,051,573); the GR antagonist compounds disclosed in U.S. Pat. Nos.
5,696,127 and 6,570,020; the GR antagonist compounds disclosed in US Patent
Application 20020077356, the glucocorticoid receptor antagonists disclosed
in Bradley et al., J. Med. Chem. 45, 2417-2424 (2002), e.g.,
octahydro-phenanthrene-2,7-diol ("CP 394531") and
tahydro-phenanthrene-2,7-diol ("CP 409069") the compounds disclosed in PCT
International Application No. WO 96/19458, which describes non-steroidal
compounds which are high-affinity, highly selective antagonists for steroid
receptors, such as 6-substituted-1,2-dihydro-N-protected-quinolines; and
some .kappa. opioid ligands, such as the .kappa. opioid compounds
xyl]benzeneacetamide), bremazocine and ethylketocyclazocine; and the
non-specific opioid receptor ligand, naloxone, as disclosed in Evans et al.,
Endocrin., 141:2294-2300 (2000).
C. Identifying Specific Glucocorticoid Receptor Antagonists
Because any specific GR antagonist can be used to treat gastroesophageal
reflux disease in a subject, in addition to the compounds and compositions
described above, additional useful GR antagonists can be determined by the
skilled artisan. A variety of such routine, well-known methods can be used
and are described in the scientific and patent literature. They include in
vitro and in vivo assays for the identification of additional GR
antagonists. A few illustrative examples are described below.
One assay that can be used to identify a GR antagonist of the invention
measures the effect of a putative GR antagonist on tyrosine amino-transferase
activity in accordance with the method of Granner, Meth. Enzymol. 15:633,
1970. This analysis is based on measurement of the activity of the liver
enzyme tyrosine amino-transferase (TAT) in cultures of rat hepatoma cells (RHC).
TAT catalyzes the first step in the metabolism of tyrosine and is induced by
glucocorticoids (cortisol) both in liver and hepatoma cells. This activity
is easily measured in cell extracts. TAT converts the amino group of
tyrosine to 2-oxoglutaric acid. P-hydroxyphenylpyruvate is also formed. It
can be converted to the more stable p- hydoxybenzaldehyde in an alkaline
solution and quantitated by absorbance at 331 nm. The putative GR antagonist
is co-administered with cortisol to whole liver, in vivo or ex vivo, or
hepatoma cells or cell extracts. A compound is identified as a GR antagonist
when its administration decreases the amount of induced TAT activity, as
compared to control (i.e., only cortisol or GR agonist added) (see also
Shirwany, Biochem. Biophys. Acta 886:162-168, 1986).
Further illustrative of the many assays which can be used to identify
compositions utilized in the methods of the invention, in addition to the
TAT assay, are assays based on glucocorticoid activities in vivo. For
example, assays that assess the ability of a putative GR antagonist to
inhibit uptake of 3H-thymidine into DNA in cells which are stimulated by
glucocorticoids can be used. Alternatively, the putative GR antagonist can
complete with 3H-dexamethasone for binding to a hepatoma tissue culture GR
(see, e.g., Choi, et al., Steroids 57:313-318, 1992). As another example,
the ability of a putative GR antagonist to block nuclear binding of
3H-dexamethasone-GR complex can be used (Alexandrova et al., J. Steroid
Biochem. Mol. Biol. 41:723-725, 1992). To further identify putative GR
antagonists, kinetic assays able to discriminate between glucocorticoid
agonists and antagonists by means of receptor-binding kinetics can also be
used (as described in Jones, Biochem J. 204:721-729, 1982).
In another illustrative example, the assay described by Daune, Molec. Pharm.
13:948-955, 1977; and in U.S. Pat. No. 4,386,085, can be used to identify
anti-glucocorticoid activity. Briefly, the thymocytes of adrenalectomized
rats are incubated in nutritive medium containing dexamethasone with the
test compound (the putative GR antagonist) at varying concentrations.
3H-uridine is added to the cell culture, which is further incubated, and the
extent of incorporation of radiolabel into polynucleotide is measured.
Glucocorticoid agonists decrease the amount of 3H-uridine incorporated.
Thus, a GR antagonist will oppose this effect.
For additional compounds that can be utilized in the methods of the
invention and methods of identifying and making such compounds, see U.S.
Pat. No.: 4,296,206 (see above); U.S. Pat. No. 4,386,085 (see above); U.S.
Pat. Nos. 4,447,424; 4,477,445; 4,519,946; 4,540,686; 4,547,493; 4,634,695;
4,634,696; 4,753,932; 4,774,236; 4,808,710; 4,814,327; 4,829,060; 4,861,763;
4,912,097; 4,921,638; 4,943,566; 4,954,490; 4,978,657; 5,006,518; 5,043,332;
5,064,822; 5,073,548; 5,089,488; 5,089,635; 5,093,507; 5,095,010; 5,095,129;
5,132,299; 5,166,146; 5,166,199; 5,173,405; 5,276,023; 5,380,839; 5,348,729;
5,426,102; 5,439,913; and 5,616,458; and WO 96/19458, which describes
non-steroidal compounds which are high-affinity, highly selective modulators
(antagonists) for steroid receptors, such as 6-substituted-1,2-dihydro N-1
The specificity of the antagonist for the GR relative to the MR can be
measured using a variety of assays known to those of skill in the art. For
example, specific antagonists can be identified by measuring the ability of
the antagonist to bind to the GR compared to the MR (see, e.g., U.S. Pat.
Nos. 5,606,021; 5,696,127; 5,215,916; 5,071,773). Such an analysis can be
performed using either direct binding assay or by assessing competitive
binding to the purified GR or MR in the presence of a known antagonist. In
an exemplary assay, cells that are stably expressing the glucocorticoid
receptor or mineralocorticoid receptor (see, e.g., U.S. Pat. No. 5,606,021)
at high levels are used as a source of purified receptor. The affinity of
the antagonist for the receptor is then directly measured. Those antagonists
that exhibit at least a 100-fold higher affinity, often 1000-fold, for the
GR relative to the MR are then selected for use in the methods of the
A GR-specific antagonist may also be defined as a compound that has the
ability to inhibit GR-mediated activities, but not MR-mediated activities.
One method of identifying such a GR-specific antagonist is to assess the
ability of an antagonist to prevent activation of reporter constructs using
transfection assays (see, e.g., Bocquel et al, J. Steroid Biochem Molec.
Biol. 45:205-215, 1993; U.S. Pat. Nos. 5,606,021, 5,929,058). In an
exemplary transfection assay, an expression plasmid encoding the receptor
and a reporter plasmid containing a reporter gene linked to
receptor-specific regulatory elements are cotransfected into suitable
receptor-negative host cells. The transfected host cells are then cultured
in the presence and absence of a hormone, such as cortisol or analog
thereof, able to activate the hormone responsive promoter/enhancer element
of the reporter plasmid. Next the transfected and cultured host cells are
monitored for induction (i.e., the presence) of the product of the reporter
gene sequence. Finally, the expression and/or steroid binding-capacity of
the hormone receptor protein (coded for by the receptor DNA sequence on the
expression plasmid and produced in the transfected and cultured host cells),
is measured by determining the activity of the reporter gene in the presence
and absence of an antagonist. The antagonist activity of a compound may be
determined in comparison to known antagonists of the GR and MR receptors
(see, e.g., U.S. Pat. No. 5,696,127). Efficacy is then reported as the
percent maximal response observed for each compound relative to a reference
antagonist compound. A GR-specific antagonist is considered to exhibit at
least a 100-fold, often 1000-fold or greater, activity towards the GR
relative to the MR.
V. Treating Gastroesophageal Reflux Disease in a Subject Using
Glucocorticoid Receptor Antagonists
Antiglucocorticoids, such as mifepristone, are formulated as pharmaceuticals
to be used in the methods of the invention to treat gastroesophageal reflux
disease in a subject. Any composition or compound that can block a
biological response associated with the binding of cortisol or a cortisol
analogue to a GR can be used as a pharmaceutical in the invention. Routine
means to determine GR antagonist drug regimens and formulations to practice
the methods of the invention are well described in the patent and scientific
literature, and some illustrative examples are set forth below.
A. Glucocorticoid Receptor Antagonists as Pharmaceutical Compositions
The GR antagonists used in the methods of the invention can be administered
by any means known in the art, e.g., parenterally, topically, orally, or by
local administration, such as by aerosol or transdermally. The methods of
the invention provide for therapeutic treatments. The GR antagonists as
pharmaceutical formulations can be administered in a variety of unit dosage
forms depending upon whether the gastroesophageal reflux disease is being
treated during an attack, the general medical condition of each patient, the
resulting preferred method of administration and the like. Details on
techniques for formulation and administration are well described in the
scientific and patent literature, see, e.g., the latest edition of
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa.
("Remington's"). Therapeutically effective amounts of glucocorticoid
blockers suitable for practice of the method of the invention will typically
range from about 0.5 to about 35 milligrams per kilogram (mg/kg). A person
of ordinary skill in the art will be able without undue experimentation,
having regard to that skill and this disclosure, to determine a
therapeutically effective amount of a particular glucocorticoid blocker
compound for practice of this invention. For example, a particular
glucocorticoid blocker may be more effective at higher or lower doses. By
evaluating a patient using the methods described herein, a skilled
practitioner will be able to determine whether a patient is responding to
treatment and will know how to adjust the dosage levels accordingly.
In general, glucocorticoid blocker compounds may be administered as
pharmaceutical compositions by any method known in the art for administering
therapeutic drugs. Compositions may take the form of tablets, pills,
capsules, semisolids, powders, sustained release formulations, solutions,
suspensions, elixirs, aerosols, or any other appropriate compositions; and
comprise at least one compound of this invention in combination with at
least one pharmaceutically acceptable excipient. Suitable excipients are
well known to persons of ordinary skill in the art, and they, and the
methods of formulating the compositions, may be found in such standard
references as Alfonso AR: Remington's Pharmaceutical Sciences, 17th ed.,
Mack Publishing Company, Easton Pa., 1985. Suitable liquid carriers,
especially for injectable solutions, include water, aqueous saline solution,
aqueous dextrose solution, and glycols.
Aqueous suspensions of the invention contain a GR antagonist in admixture
with excipients suitable for the manufacture of aqueous suspensions. Such
excipients include a suspending agent, such as sodium carboxymethylcellulose,
methylcellulose, hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or
wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a
condensation product of an alkylene oxide with a fatty acid (e.g.,
polyoxyethylene stearate), a condensation product of ethylene oxide with a
long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a
condensation product of ethylene oxide with a partial ester derived from a
fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a
condensation product of ethylene oxide with a partial ester derived from
fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).
The aqueous suspension can also contain one or more preservatives such as
ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as sucrose,
aspartame or saccharin. Formulations can be adjusted for osmolarity.
Oil suspensions can be formulated by suspending a GR antagonist in a
vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or
in a mineral oil such as liquid paraffin; or a mixture of these. The oil
suspensions can contain a thickening agent, such as beeswax, hard paraffin
or cetyl alcohol. Sweetening agents can be added to provide a palatable oral
preparation, such as glycerol, sorbitol or sucrose. These formulations can
be preserved by the addition of an antioxidant such as ascorbic acid. As an
example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther.
281:93-102, 1997. The pharmaceutical formulations of the invention can also
be in the form of oil-in-water emulsions. The oily phase can be a vegetable
oil or a mineral oil, described above, or a mixture of these. Suitable
emulsifying agents include naturally-occurring gums, such as gum acacia and
gum tragacanth, naturally occurring phosphatides, such as soybean lecithin,
esters or partial esters derived from fatty acids and hexitol anhydrides,
such as sorbitan mono-oleate, and condensation products of these partial
esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
The emulsion can also contain sweetening agents and flavoring agents, as in
the formulation of syrups and elixirs. Such formulations can also contain a
demulcent, a preservative, or a coloring agent.
Glucocorticoid blocker pharmaceutical formulations can be prepared according
to any method known to the art for the manufacture of pharmaceuticals. Such
drugs can contain sweetening agents, flavoring agents, coloring agents and
preserving agents. Any glucocorticoid blocker formulation can be admixtured
with nontoxic pharmaceutically acceptable excipients which are suitable for
In one aspect, glucocorticoid blocker compounds suitable for use in the
practice of this invention can be administered orally. The amount of a
compound of this invention in the composition may vary widely depending on
the type of composition, size of a unit dosage, kind of excipients, and
other factors well known to those of ordinary skill in the art. In general,
the final composition may comprise from 0.000001 percent by weight (% w) to
10% w of the glucocorticoid blocker compounds, preferably 0.00001% w to 1%
w, with the remainder being the excipient or excipients. For example, the GR
antagonist mifepristone is given orally in tablet form, with dosages in the
range of between about 0.5 and 35 mg/kg, in other embodiments, dosages may
range between about 0.75 mg/kg and 15 mg/kg, or may be about 10 mg/kg.
Pharmaceutical formulations for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages
suitable for oral administration. Such carriers enable the pharmaceutical
formulations to be formulated in unit dosage forms as tablets, pills,
powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,
suspensions, etc. suitable for ingestion by the patient. Pharmaceutical
preparations for oral use can be obtained through combination of
glucocorticoid blocker compounds with a solid excipient, optionally grinding
a resulting mixture, and processing the mixture of granules, after adding
suitable additional compounds, if desired, to obtain tablets or dragee
cores. Suitable solid excipients are carbohydrate or protein fillers and
include, but are not limited to sugars, including lactose, sucrose, mannitol,
or sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose or sodium
carboxymethylcellulose; and gums including arabic and tragacanth; as well as
proteins such as gelatin and collagen. If desired, disintegrating or
solubilizing agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
In another aspect, the GR antagonists of this invention can also be
administered in the form of suppositories for rectal administration of the
drug. These formulations can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures but liquid
at the rectal temperatures and will therefore melt in the rectum to release
the drug. Such materials are cocoa butter and polyethylene glycols.
In another aspect, the GR antagonists of this invention can be administered
by in intranasal, intraocular, intravaginal, and intrarectal routes
including suppositories, insufflation, powders and aerosol formulations (for
examples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol.
35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995).
In another aspect, the GR antagonists of the invention can also be delivered
transdermally, by a topical route, formulated as applicator sticks,
solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies,
paints, powders, and aerosols.
In still another aspect, the GR antagonists of the invention can also be
delivered as microspheres for slow release in the body. For example,
microspheres can be administered via intradermal injection of drug (e.g.,
mifepristone)-containing microspheres, which slowly release subcutaneously
(see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and
injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995);
or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm.
Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routes afford
constant delivery for weeks or months.
The GR antagonist pharmaceutical formulations of the invention can be
provided as a salt and can be formed with many acids, including but not
limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic solvents
that are the corresponding free base forms. In other cases, the preferred
preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%
sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with
buffer prior to use.
In one embodiment, the GR antagonist formulations of the invention are
useful for parenteral administration, such as intravenous (IV)
administration. The formulations for administration will commonly comprise a
solution of the GR antagonist (e.g., mifepristone) dissolved in a
pharmaceutically acceptable carrier. Among the acceptable vehicles and
solvents that can be employed are water and Ringer's solution, an isotonic
sodium chloride. In addition, sterile fixed oils can conventionally be
employed as a solvent or suspending medium. For this purpose any bland fixed
oil can be employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid can likewise be used in the preparation of
injectables. These solutions are sterile and generally free of undesirable
matter. These formulations may be sterilized by conventional, well known
sterilization techniques. The formulations may contain pharmaceutically
acceptable auxiliary substances as required to approximate physiological
conditions such as pH adjusting and buffering agents, toxicity adjusting
agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium lactate and the like. The concentration of GR antagonist in
these formulations can vary widely, and will be selected primarily based on
fluid volumes, viscosities, body weight, and the like, in accordance with
the particular mode of administration selected and the patient's needs. For
IV administration, the formulation can be a sterile injectable preparation,
such as a sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents. The sterile injectable
preparation can also be a sterile injectable solution or suspension in a
nontoxic parenterally-acceptable diluent or solvent, such as a solution of
In another embodiment, the GR antagonist formulations of the invention can
be delivered by the use of liposomes which fuse with the cellular membrane
or are endocytosed, i.e., by employing ligands attached to the liposome, or
attached directly to the oligonucleotide, that bind to surface membrane
protein receptors of the cell resulting in endocytosis. By using liposomes,
particularly where the liposome surface carries ligands specific for target
cells, or are otherwise preferentially directed to a specific organ, one can
focus the delivery of the GR antagonist into the target cells in vivo. (See,
e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin.
Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).
B. Determining Dosing Regimens for Glucocorticoid Receptor Antagonists
The methods of this invention treat gastroesophageal reflux disease in a
subject. The amount of GR antagonist adequate to accomplish this is defined
as a "therapeutically effective dose". The dosage schedule and amounts
effective for this use, i.e., the "dosing regimen," will depend upon a
variety of factors, including the severity of the gastroesophageal reflux
disease, whether the treatment is being given during the course of a
gastroesophageal reflux disease attack, or prophylactically, the patient's
physical status, age, gastroesophageal reflux disease history, and the like.
In calculating the dosage regimen for a patient, the mode of administration
also is taken into consideration.
The dosage regimen also takes into consideration pharmacokinetics parameters
well known in the art, i.e., the GR antagonists' rate of absorption,
bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones,
J. Steroid Biochem. Mol. Biol. 58:611-617, 1996; Groning, Pharmazie
51:337-341, 1996; Fotherby, Contraception 54:59-69, 1996; Johnson, J. Pharm.
Sci. 84:1144-1146, 1995; Rohatagi, Pharmazie 50:610-613, 1995; Brophy, Eur.
J. Clin. Pharmacol. 24:103-108, 1983; the latest Remington's, supra). For
example, in one study, less than 0.5% of the daily dose of mifepristone was
excreted in the urine; the drug bound extensively to circulating albumin
(see Kawai, supra, 1989). The state of the art allows the clinician to
determine the dosage regimen for each individual patient, GR antagonist and
disease or condition treated. As an illustrative example, the guidelines
provided below for mifepristone can be used as guidance to determine the
dosage regiment, i.e., dose schedule and dosage levels, of any GR antagonist
administered when practicing the methods of the invention.
Single or multiple administrations of GR antagonist formulations can be
administered depending on the dosage and frequency as required and tolerated
by the patient. The formulations should provide a sufficient quantity of
active agent, i.e., mifepristone, to effectively treat gastroesophageal
reflux disease in a subject. Thus, one typical pharmaceutical formulations
for oral administration of mifepristone is in a daily amount of between
about 0.5 to about 35 mg per kilogram of body weight per day. In an
alternative embodiment, dosages are from about 5 mg to about 15 mg per kg of
body weight per patient per day are used in the practice of the invention.
Lower dosages can be used, particularly when the drug is administered to an
anatomically secluded site, such as the cerebral spinal fluid (CSF) space,
in contrast to administration orally, into the blood stream, into a body
cavity or into a lumen of an organ. Substantially higher dosages can be used
in topical administration. Actual methods for preparing parenterally
administrable GR antagonist formulations will be known or apparent to those
skilled in the art and are described in more detail in such publications as
Remington's, supra. See also Nieman, In "Receptor Mediated Antisteroid
Action," Agarwal, et al., eds., De Gruyter, New York, 1987.
After a pharmaceutical comprising a GR antagonist of the invention has been
formulated in a acceptable carrier, it can be placed in an appropriate
container and labeled for treatment of an indicated condition. For
administration of GR antagonists, such labeling would include, e.g.,
instructions concerning the amount, frequency and method of administration.
In one embodiment, the invention provides for a kit for treating
gastroesophageal reflux disease in a subject which includes a GR antagonist
and instructional material teaching the indications, dosage and schedule of
administration of the GR antagonist.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in
light thereof will be suggested to persons skilled in the art and are to be
included within the spirit and purview of this application and scope of the
Claim 1 of 14 Claims
1. A method of treating gastroesophageal
reflux disease in a subject in need thereof; the method comprising the
step of administering to the subject an effective amount of a specific
glucocorticoid receptor antagonist to treat gastroesophageal reflux
disease in the subject, with the proviso that the subject is not otherwise
in need of treatment with a glucocorticoid receptor antagonist.
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