Note: Descriptions are shown in the official language in which they were submitted.
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A NOVEL CRYSTALLINE FORM OF 6-HYDROXY
3-(4-[2-(PIPERIDIN-1-YL)ETHOXY]
PHENOXY)-2-(4-METHOXYPHENYL)BENZO[b]THIOPHENE HYDROCHLORIDE
Background of the Invention
6-Hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-
methoxyphenyl)benzo[b]thiophene hydrochloride (arzoxifene)
was first described generically in U.S. Patent No. 5,510,357
and was specifically disclosed in U.S. Patent No. 5,723,474
('474) and European Patent Application 0729956. Arzoxifene
is a nonsteroidal mixed estrogen antagonist/agonist, useful
for, inter alia, lowering serum cholesterol and for
inhibiting hyperlipidemia, osteoporosis, estrogen dependent
cancers including breast and uterine cancer, endometriosis,
CNS disorders including Alzheimer's disease, aortal smooth
muscle cell proliferation, and restenosis.
Specifically, arzoxifene is useful for, and is being
clinically evaluated for the treatment of receptor positive
metastatic breast cancer; the adjuvent treatment of receptor
positive patients following appropriate systemic or local
therapy; the reduction of recurrence of invasive and
noninvasive breast cancer; and the reduction of the
incidence of invasive breast cancer and ductal carcinoma in
situ (DCIS). Arzoxifene is also useful in combination with
radiotherapy, aromatase inhibitors, LHRH analogues, and
acetyl choline esterase (AChE) inhibitors.
X-ray powder diffraction (XRD), thermogravimetric
(TGA), proton nuclear magnetic resonance (1H NMR) and Karl
Fischer (KF) analyses of bulk arzoxifene isolated by the
procedures taught in '474 later indicated that said material
was hydrated, poorly crystalline, and contained variable
amounts of an organic volatile (ethyl acetate) in its
lattice.
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Poorly crystalline materials are typically less
desirable than highly crystalline materials for formulation
processing. In addition, it is generally not desirable to
formulate pharmaceuticals containing substantial amounts of
organic solvent (e. g., ethyl acetate) due to potential
solvent toxicity to the recipient thereof and changes in
potency of the pharmaceutical as a function of the solvent.
Although the arzoxifene prepared by the procedures
taught in '474 could be used as a pharmaceutical, it would
be highly desired and advantageous to find a more
crystalline form of arzoxifene that did not contain an
organic solvent within its crystal lattice which could be
reproducibly and efficiently prepared on a commercial scale.
Summary of the Invention
The present invention is related to a novel non
stoichiometric hydrated crystalline form of 6-hydroxy-3-(4
[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-
methoxyphenyl)benzo[b]thiophene hydrochloride (F-I) having
an X-ray diffraction pattern which comprises the following
peaks: 7.9 ~0.2, 10.7 ~0.2, 14.9 ~0.2, 15.9 ~0.2, 18.3 ~0.2,
and 20.6 ~0.2o in 28; when obtained from a copper radiation
source.
Moreover the present invention relates to a
pharmaceutical formulation comprising F-I; one or more
pharmaceutical carriers, diluents, or excipients; and
optionally estrogen, optionally progestin, optionally an
aromatase inhibitor, optionally an LHRH analogue and
optionally an acetyl choline esterase (AChE) inhibitor.
In addition, the present invention is related to
methods for using F-I to inhibit pathological conditions
such as: uterine fibrosis, endometriosis, aortal smooth
muscle cell proliferation, restenosis, breast cancer,
uterine cancer, prostatic cancer, benign prostatic
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hyperplasia, bone loss, osteoporosis, cardiovascular
disease, hyperlipidemia, CNS disorders, and Alzheimer's
disease and for using F-I for the manufacture of a
medicament for inhibiting same.
The present invention is further related to methods for
using F-I to up-regulate choline acetyltransferase (ChAT)
and for using F-I for the manufacture of a medicament for
up-regulating same.
Brief Description of the Figures
Figure 1 is a representative differential scanning
calorimetry (DSC)/TGA trace of S-II.
Figure 2 is a representative DSC/TGA trace of F-I.
Figure 3 is a representative DSC/TGA trace of F-III.
Figure 4 depicts moisture sorption isotherms for F-I
and F-III.
Figure 5 depicts desolvation of S-II as a function of
drying time and temperature.
Detailed Description of the Invention
Bulk arzoxifene prepared by the procedure taught in
'474 (Example 41, crystallization from a mixture of ethanol
and ethyl acetate, filtration and drying of the filter cake
in vacuo to a constant weight at room temperature) was
characterized by XRD and was found to be poorly crystalline.
1H NMR confirmed that the bulk material contained 6~ ethyl
acetate.
The crystallization procedure taught in '474 was
subsequently modified so that ethanol was added to a
suspension of crude arzoxifene in refluxing ethyl acetate.
Upon cooling and vacuum filtration, the solid that results
from this modified procedure is a highly crystalline mixed
ethyl acetate/water solvate of arzoxifene (hereinafter
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referred to as S-II) which was later discovered to be a
starting material for F-I.
F-I may be prepared by removing the ethyl acetate from
S-II's crystal lattice by vacuum drying/annealing S-II at
elevated temperatures. The time and temperature required to
anneal S-II in order to prepare F-I will vary from lot to
lot but is typically on the order of 5 days at around 100oC.
High temperatures are needed to effect the conversion of S-
II to F-I via this procedure, since slurrying S-II in water
at ambient temperature or storing a sample at 98~ RH for 3
weeks afforded no conversion to F-I. Furthermore, drying S-
II in a convection oven at high temperatures did not de-
solvate the material either, suggesting that a vacuum is
also required to pull the ethyl acetate from S-II's lattice.
Preferably, F-I is readily prepared and isolated at
ambient temperature by crystallization of arzoxifene (or any
polymorph/solvate thereof) from tetrahydrofuran. This
crystallization is preferably performed by initially
dissolving arzoxifene in wet tetrahydrofuran (1-10~ water by
volume, preferably 2.5-7.5~ and most preferably 4.5 to 5.5~)
followed by removal of said water via atmospheric
distillation. An example of this crystallization is
detailed below in Example 2. lr~hen F-I is prepared via this
improved crystallization procedure, a total related
substance (TRS) level of <0.5 ~ can be expected.
Suitable arzoxifene starting material for this
crystallization includes, but is not limited to, S-II, F-
III, arzoxifene prepared by the procedures taught in '474,
or any mixture thereof. It is not important which form of
arzoxifene one starts with because crystallization from
tetrahydrofuran, according to the procedures described
herein, results in F-I crystals. For commercial scale
synthesis of F-I, it may be advantageous to seed the
crystallization with F-I.
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k,
F-III, another non-stoichiometric hydrate of
arzoxifene, is readily prepared and isolated at ambient
temperature by crystallization of arzoxifene (or any
polymorph/solvate thereof) from a mixture of isopropyl
alcohol (IPA) and water. The ratio of water to IPA (v:v) is
generally about 1:1 to 9:1. More preferably, the ratio is
between 2.5 and 5.6:1. Most preferably, the ratio is
between 3 to 5.6:1. The ratio of IPA to water is not
critical to effect crystallization of F-III but does affect
the yield. For commercial scale synthesis of F-III, it may
be advantageous to seed the crystallization with F-III.
Suitable arzoxifene starting material for the above
crystallization include, but are not limited to, S-II, F-I,
arzoxifene prepared by the procedures taught in '474, or any
mixture thereof.
Characterization and Differentiation of S-II, F-I and F-III
DSC/TGA and XRD methods were used to characterize S-II,
F-I and F-III. TGA is often very useful for distinguishing
between different solid forms of a material because the
temperatures) at which a physical change in a material
occurs is usually characteristic of the polymorph or
solvate. DSC is a technique that is often used to screen
compounds for polymorphism and solvate formation. Lastly,
XRD is a technique that detects long-range order in a
crystalline material.
Arzoxifene prepared by the procedures taught in '474
gave XRD patterns with poor signal-to-noise ratios and a
raised baseline, indicative of poorly crystalline material.
Therefore, comparisons of F-I and F-III are made to the
material (S-II) produced by the modified arzoxifene
crystallization procedure discussed above (addition of
ethanol to a suspension of arzoxifene in refluxing ethyl
acetate).
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Representative DSC/TGA traces of S-II, F-I and F-III
are shown in Figures 1, 2 and 3, respectively. The DSC
trace for S-II shows a broad endotherm beginning at about
62oC, corresponding to the loss of ethyl acetate and water
from the lattice. The endotherm beginning at about 152oC
represents a melt. The TGA weight loss of approximately
2.5~ occurs simultaneous with the first transition, while
the remaining 0.5~ weight loss occurs up to the onset of
melting, suggesting that some solvent molecules are more
tightly held in the lattice.
The DSC trace of F-I shows a broad endotherm beginning
at about 75oC, followed by a second endotherm beginning at
about 155°C corresponding to a melt. The TGA trace of F-I
shows a gradual weight loss of 0.3~ followed by a sharp loss
of 1.5~, which together represent dehydration of the
lattice. The onset of the first DSC transition and the
corresponding TGA weight loss are offset slightly due to the
difference in heating rates. The initial weight loss
represents weakly held waters of hydration while the second
weight loss is consistent with approximately 0.5 mole of
water present in the lattice at very low relative humidities
(below 5~ - see moisture sorption data).
The DSC trace of F-III features a broad, low
temperature endotherrn at about 30°C, followed by a second
broad and relatively weak endotherm beginning at about 70oC,
and a final transition beginning at about 146oC
corresponding to a melt. The sharp 1.5~ (--0.5 mole) weight
loss in the TGA coincident with the first endotherm
corresponds to loss of weakly held water molecules, while
the additional --1.6~ weight loss above 60oC represents loss
of more tightly held water molecules, i.e., those which are
present at very low relative humidities. The weight loss
observed after 170oC corresponds to decomposition of F-III.
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The XRD patterns of F-I and F-III feature sharp peaks
and a flat baseline, indicative of highly crystalline
materials. The angular peak positions in 2A and
corresponding I/Io data for representative samples of F-I,
F-III and S-II is tabulated in Table 1. Although many of
the intense reflections are generally at similar diffraction
angles, each of the forms gives a different powder pattern,
allowing for a clear distinction between S-II, F-I and F-
III.
It is well known in the crystallography art that, for
any given polymorph, the relative intensities of the
diffraction peaks may vary due to preferred orientation
resulting from factors such as crystal morphology. Where
the effects of preferred orientation are present, peak
intensities are altered, but the characteristic peak
positions of the polymorph are unchanged. See, e.g., The
United States Pharmacopeia #23, National Formulary #18,
pages 1843-1844, 1995. Thus, based on peak intensities as
well as peak position, F-I may be identified by the presence
of peaks at 7.9 ~0.2, 10.7 ~0.2, 14.9 ~0.2, 15.9 ~0.2, 18.3
~0.2, and 20.6 ~0.2o in 2A; when the pattern is obtained
from a copper radiation source.
Table 1
S-II F-I F-III
~
2~ () I/Ip (%) 28 () I/Io (%) 2e () I/Io (%)
4.67 1.3 4.92 2.6 4.63 20.8
5.03 6 7.69 34.6 7.82 100
6.83 5.8 7.91 100 9.29 16.9
7.17 16.1 9.89 2.5 10.16 22.7
7.73 100 10.22 2 10.35 5.4
9.03 1.3 10.74 7.4 13.77 10.7
9.31 1.7 14.86 9.1 13.97 15.2
9.66 2.4 15.45 2.3 15.06 6.9
10.27 1.6 15.92 15.9 15.71 22.3
10.47 2.2 16.67 1.7 15.87 7.4
10.91 6.3 16.98 3.1 16.35 34.5
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Table 1 (continued
S-II F-I I
Ze c~~ =i~o (%~ ae c~~ =i=o c~~ Zg ~o~ zi=o c~~
13.63 2.1 18.28 17.8 16.77 12.3
14.09 4.6 18.56 7 17.28 10
15.10 4.1 20.58 13.1 17.62 47.9
15.52 10.5 20.85 8.8 18.09 43.9
16.45 9.1 21.64 3.9 20.43 42
16.67 7.6 22.19 4.8 20.80 33.6
17.21 4.9 22.65 2.9 21.31 42.7
17.53 2.4 23.28 3.4 21.71 13
18.33 28.2 23.97 11.8 21.85 14.5
18.69 11.1 24.31 6.3 22.13 12.8
19.37 3.5 25.52 3.9 22.26 16.3
20.29 8.6 26.20 3.4 23.51 13.2
20.64 17.2 26.47 3.1 23.69 15.9
21.02 12.7 28.84 6.4 23.91 25.6
21.68 5.1 30.13 3.5 24.31 38.7
22.01 8.3 31.12 2.9 25.22 8
22.29 8 25.67 8.9
23.17 7.8 27.05 18.9
23.39 9.1 27.89 13.3
24.30 13.6 28.24 8.6
25.76 3.4 28.71 21.3
28.10 1.8 29.89 8.9
28.73 10.9 30.24 18.7
29.42 3.2 30.88 5.8
30.00 3.7 31.44 7.6
30.89 2.1 33.06 4.5
31.34 2.4 34.36 6
31.70 1.1
32.81 1
32.91 0.8
33.48 2
26.05 4
26.63 5.5
27.01 3.1
27.49 2.8
Further Characterization of F-I and F-III
Hygroscopicicity studies were performed on F-I and F-
III. The moisture sorption isotherms for F-I and F-III are
shown in Figure 4. Upon initial exposure of the samples to
approximately 5~ RH, there was an immediate weight gain of
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1.5~ and 1.7~ moisture for F-I and F-III, respectively,
equivalent to approximately 0.5 mole of water. Both forms
show a continuous sorption of moisture through the entire
humidity range, which is likely due to incorporation of
water molecules in the lattices.
The difference in the moisture uptake of the two forms
likely reflects the amount of water that can be incorporated
into the two lattices (i.e., the amount of available space
in the lattice that can accommodate water molecules). Lack
of hysteresis in the sorption-desorption isotherms of F-I
and F-III indicates that the crystal forms rapidly
equilibrate at any given humidity.
The moisture sorption profiles for F-I and F-III reveal
that these forms are essentially non-stoichiometric
hydrates. At ambient relative humidity (about 50~ RH), F-I
contains approximately 1.7$ water, corresponding to 0.5
moles of water, while F-III has sorbed about 3.0~ water
which corresponds to about 0.85 moles of water.. The bulk
forms of F-I and F-III rapidly equilibrate with the
atmosphere, so that the water content observed by analytical
techniques is a reflection of the relative humidity at the
time of data collection. Lot-to-lot differences observed in
the DSC data likely results from the samples being hydrated
to different extents due to different ambient storage
conditions.
XRD patterns were obtained for samples of F-I and F-III
stored at different relative humidities (0, 22, 50, and
80~). There is a gradual shifting of the initial (0~ RH) F-
III peaks at about 13.8, 17.6, 18.0, 20.5 and 24.0° in 28 as
well as slight shifting of less intense peaks, as the
relative humidity is increased. These observed changes in
the XRD patterns of F-III indicate that the unit cell
dimensions are changing, presumably to accommodate weakly
held water molecules as the relative humidity is increased.
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The continuous shifting of peaks with humidity correlates
well with moisture sorption data that showed a gradual
weight gain over this RH range, providing evidence for
variable hydrate formation.
A similar experiment was carried out on F-I to
determine whether varying the relative humidity would have a
similar effect on its lattice (0, 25, 52, 73 and 95~ RH).
Very slight shifting of the 0~ RH peaks at about 7.7, 18.3,
18.5, 20.5, 20.8° in 28 is observed as the relative humidity
is increased. The peaks at about 7.7, 20.8, and 24.1 also
appear to become slightly broadened and less resolved at
higher relative humidities, indicating that water is being
sorbed into amorphous components (or plasticizes the solid),
particularly at 73 and 95~ RH. The shifting of peaks in the
XRD patterns of F-I is less dramatic than the peak shifts
observed as F-III was exposed to different relative
humidities. This suggests that the F-I lattice does not
undergo the same expansion and/or contraction as the F-III
lattice.
F-I and F-III were found to be stable over the entire
relative humidity range, despite the ability of F-III to
sorb nearly twice as much water. The two forms were found
to have comparable crystal size, morphology, aqueous
solubilities and dissolution rates.
A drying study was carried out to monitor the
desolvation of S-II as a function of drying time and
temperature (see Figure 5). XRD patterns were taken at
various timepoints during the desolvation experiment. Many
diffraction peaks from the desolvation study of S-II appear
at similar angles to F-I, confirming that the lattices of S-
II and F-I are very similar. The disappearance of
diffraction peaks at about 6.8, 7.2 and l4.Oo in 2A after
only minimal drying suggests that these reflections may be
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attributed to crystallographic planes containing partial
electron density of ethyl acetate molecules.
Extended annealing of the solvated material under
vacuum at high temperatures yielded F-I. F-I prepared this
way showed a high degree of crystallinity by XRD.
Therefore, material generated by crystallization from a
solution of ethanol and ethyl acetate followed by vacuum
drying for only a few hours as taught in '474 showed very
poor crystallinity because such a procedure results in
partially desolvated S-II.
F-I and F-III have several advantages over the prior
art form of arzoxifene described above. Relative to the
arzoxifene produced by the procedures taught in '474, F-I
and F-III are more stable at ambient temperature and are,
therefore, more amenable to pharmaceutical development,
i.e., development of a dosage formulation. In addition, F-I
and F-III are much more crystalline than the form disclosed
in '474. Crystalline materials are generally less
hygroscopic and more stable (e. g., less prone to chemical
degradation, maintains consistent potency) than amorphous
materials and are, therefore, more desirable for formulation
processing. Furthermore, unlike the form of arzoxifene
produced by the procedures taught in '474, which contained
ethyl acetate and water in its lattice, F-I and F-III
contain only water.
Characterization Methods
DSC measurements were performed on a TA Instruments
2920 Modulated DSC attached to a Thermal Analyst 3100 and
equipped with a refrigerated cooling system. Samples (3-5
mg) were heated in crimped aluminum pans from 10 to 240oC at
a heating rate of 2oC/min.
TGA analyses were performed on a TA Instruments 2050
Thermogravimetric Analyzer attached to a Thermal Analyst
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3100. Samples (5-10 mg) were heated in open pans from 25oC
to 250oC at a heating rate of 5oC/min.
XRD patterns were obtained on a Siemens D5000 X-ray
powder diffractometer, equipped with a CuKOC source (h =
1.54056 ~) and a Kevex solid-state detector, operating at 50
kV and 40 mA. Each sample was scanned between 4o and 35o in
2A. Samples were allowed to equilibrate for at least 30
minutes at the desired-temperature and/or relative humidity
before data collection.
Hygroscopicity measurements were made for F-I and F-III
using the VTI method as follows. Each sample was dried
under vacuum at 60oC until no further weight loss was
detected, at which time the sample chamber was brought to 0~
relative humidity. Moisture sorption isotherms were
obtained at 25oC using a VTI vacuum moisture balance with
the following conditions: sample size 10-15 mg,
adsorption/desorption range 0-95~ relative humidity, step
interval 5~, sample interval 10 minutes.
The following examples further illustrate processes for
preparing the hydrate of the present invention. The
examples are not intended to be limiting to the scope of
these processes in any respect, and should not be so
construed.
2 5 Preparatioris
Preparation 1
S-II
Crude arzoxifene (1.58 g of material prepared by the
procedure of Example 41 in U.S. Patent No. 5,723,474, the
teachings of which are herein incorporated by reference) was
suspended in 28 mL ethyl acetate and heated to reflux.
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Ethanol (18 mL) was added to effect dissolution. The
solution was maintained at reflux for 20 minutes and then
allowed to cool to room temperature. The precipitate was
isolated by vacuum filtration and was washed with 30 mL
ethyl acetate to give 1.05 g of a powdery, white solid.
Examples
Example 1
F-I from S-II
S-II was dried in a vacuum oven (-25 in. Hg) at 100oC
for 118 hours to yield F-I.
Example 2
Improved Procedure for Preparing F-I from Arzoxifene
A 1L, 3-necked round bottom flask equipped with a
reflux condenser and an overhead agitator is charged with
25.0 g of arzoxifene, 475 ml of tetrahydrofuran and 25 ml of
water. The reaction vessel is then equipped for simple
distillation. The reaction mixture is heated to reflux and
250 ml of distillate are removed. Heat is briefly removed
and 250 ml of fresh anhydrous tetrahydrofuran is added to
the vessel. Atmospheric distillation is continued with
removal of an additional 250 ml of distillate. Heat is
briefly removed, 250 ml of fresh tetrahydrofuran added, and
an additional 250 ml of distillate are removed. An
additional 250 ml of tetrahydrofuran is added, and the
reaction mixture is held at reflux. VAith this
tetrahydrofuran addition, a white precipitate forms. The
agitated reaction mixture is allowed to cool slowly over 3
hours during which time additional solids precipitate and
the slurry reached ambient temperature. The crystalline
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slurry is filtered and vacuum dried at 50°C for forty eight
hours with a slight NZ purge. Yield 22.50 g (90.00 . XRD
analysis showed the spectrum of the wet cake and the dry
solid are substantially identical, and substantially
identical to that of F-I previously prepared. DSC analysis
afforded a melting point of 157°C while TGA analysis showed
a 1.5~ mass loss between ambient temperature and 100°C. The
HPLC purity calculated as the free base was 88.1 vs, a
theoretical potency of 92.9. HPLC analysis showed a total
related substance level of 0.44.
Example 3
F-I from [6-Benzyloxy-3-[4-[2-(piperidin-1-
yl)ethoxy)phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiopene-(S-
oxide )
Tetrahydrofuran (261 ml), water (45 ml) concentrated
sulfuric acid (6.14 g) and [6-benzyloxy-3-[4-[2-(piperidin-
1-yl)ethoxy)phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiopene-
(S-oxide) (HPLC potency 99~, HPLC total related substance
level 0.350 were combined and stirred until homogeneous.
10~ Pd/C (5.6 g slurried in 22 ml of water) was added with a
5 ml water rinse. The resulting slurry was evacuated and
overlaid with 60 psi of hydrogen. The reaction temperature
was adjusted to 30°C. After 2 hours, 10~ Pd/C (5.6 g) of
was added with water (30 ml). Hydrogenation at 60 psi and
30°C was continued for an additional 22 hours. An
additional 4.40 g of 10~ Pd/C in 30 ml water was added and
hydrogenation at 60 psi and 30°C continued for an additional
2.5 hours. The catalyst was removed by filtration and the
pH of the filtrate was adjusted to 7.24 with 50~ sodium
hydroxide. Sodium chloride (8.66 g) dissolved in water (18
ml) was added and the biphasic solution stirred for 30
minutes. The phases were separated and the aqueous phase
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was back extracted with 50 ml of tetrahydrofuran. The
organic phases were combined and concentrated by atmospheric
distillation to a volume of 50 ml. To the concentrate at
24°C was added methanol, 180 ml over a 1 hour period. The
resulting crystalline slurry was stirred for 30 minutes at
24°C, cooled to 0°C and stirred for 1 hour. The solids were
isolated by filtration and washed sequentially with 39 ml of
water and 39 ml of methanol followed by vacuum drying
overnight at 50°C. Yield 15.52 g (67.8$).
A portion of the product from above (10 g) are
recrystallized from tetrahydrofuran and water as described
in Example 2.
Utilities
As used herein, the term "effective amount" means an
amount of F-I that is capable of inhibiting conditions, or
detrimental effects thereof, described herein. When F-I is
co-administered with estrogen, progestin, an aromatase
inhibitor, an LHRH analogue, or an AChE inhibitor, the term
"effective amount" also means an amount of such an agent
capable of producing its intended effect.
The terms "inhibiting" and "inhibit" include their
generally accepted meaning, i.e., preventing, prohibiting,
restraining, alleviating, ameliorating, slowing, stopping,
or reversing the progression or severity of a pathological
condition, or sequela thereof, described herein.
The terms "preventing", "prevention of", "prophylaxis",
"prophylactic" and "prevent" are used herein interchangeably
and refer to reducing the likelihood that the recipient of
F-I will incur or develop any of the pathological
conditions, or sequela thereof, described herein.
The terms "estrogen deprived" and "estrogen
deprivation" refer to a condition, either naturally
occurring or clinically induced, where a woman can not
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produce sufficient endogenous estrogenic hormones to
maintain estrogen dependent functions, e.g., menses,
homeostasis of bone mass, neuronal function, cardiovascular
condition, etc. Such estrogen deprived situations arise
from, but are not limited to, menopause and surgical or
chemical ovarectomy, including its functional equivalent,
e.g., medication with an aromatase inhibitor, GnRH agonists
or antagonists, ICI 182780, and the like. Disease states
associated with an estrogen deprived state include, but are
not limited to: bone loss, osteoporosis, cardiovascular
disease and hyperlipidemia.
As used herein, the term "estrogen" includes steroidal
compounds having estrogenic activity such as, for example,
17~-estradiol, estrone, conjugated estrogen (Premarin~),
equine estrogen 17R-ethynyl estradiol, and the like. A
preferred estrogen-based compound is Premarin~, and
norethylnodrel.
As used herein, the term "progestin" includes compounds
having progestational activity such as, for example,
progesterone, norethylnodrel, nongestrel, megestrol acetate,
norethindrone, and the like. Norethindrone is a preferred
progestin-based agent.
As used herein the term "aromatase inhibitor" includes
compounds capable of inhibiting aromatase, for example
commercially available inhibitors such as aminoglutemide
(CYTANDREN~), Anastrazole (ARIMIDEX~), Letrozole (FEMARA~),
Formestane (LENATRON~), Exemestane (AROMASIN~), and the
like.
As used herein, the term "LHRH analogue" refers to an
analogue of lutenizing hormone releasing hormone that
inhibits estrogen production in a premenopausal women
including for example, goserlin (ZOLADEX~), leuprolide
(LUPRON~) and the like.
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As used herein, the term "AChE inhibitor" includes
compounds that inhibit acetyl choline esterase, for example,
physostigmine salicylate, tacrine hydrochloride, donepezil
hydrochloride and the like.
The term "up-regulate ChAT" refers to increasing the
enzymatic activity of ChAT, i.e., promoting the conversion
of choline to acetyl choline. This promotion would include
an increase in the efficiency and/or rate of reaction of
ChAT and choline and/or an increase in the amount of ChAT
present at the site of action. This increase in the amount
of enzyme present may be due to gene regulation or other
synthetic step of the enzyme's formation and/or a decrease
in the enzyme's de-activation and metabolism.
Selected Testing Procedures
General Rat Preparation Procedure: Seventy-five day
old (unless otherwise indicated) female Sprague Dawley rats
(weight range of 200 to 225g) are obtained from Charles
River Laboratories (Portage, MI). The animals are either
bilaterally ovariectomized (OVX) or exposed to a Sham
surgical procedure at Charles River Laboratories, and then
shipped after one week. Upon arrival, they are housed in
metal hanging cages in groups of 3 or 4 per cage and have ad
Iibitum access to food (calcium content approximately 0.5~)
and water for one week. Room temperature is maintained at
22.20 ~ 1.70C with a minimum relative humidity of 40~. The
photoperiod in the room was 12 hours light and 12 hours
dark.
Dosing Regimen Tissue Collection: After a one week
acclimation period (therefore, two weeks post-OVX) daily
dosing with F-I is initiated. 17a-ethynyl estradiol or F-I
is given orally, unless otherwise stated, as a suspension in
1~ carboxymethylcellulose or dissolved in 20~ cyclodextrin.
Animals are dosed daily for 4 days. Following the dosing
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regimen, animals are weighed and anesthetized with a
ketamine: Xylazine (2:1, v:v) mixture and a blood sample is
collected by cardiac puncture. The animals are then
sacrificed by asphyxiation with C02, the uterus is removed
through a midline incision, and a wet uterine weight is
determined. 17a-ethynyl estradiol is obtained from Sigma
Chemical Co., St. Louis, MO.
Cardiovascular Disease/Hyperlipidemia
The blood samples from above are allowed to clot at
room temperature for 2 hours, and serum is obtained
following centrifugation for 10 minutes at 3000 rpm. Serum
cholesterol is determined using a Boehringer Mannheim
Diagnostics high performance cholesterol assay. Briefly the
cholesterol is oxidized to cholest-4-en-3-one and hydrogen
peroxide. The hydrogen peroxide is then reacted with phenol
and 4-aminophenazone in the presence of peroxidase to
produce a p-quinone imine dye, which .is read
spectrophotemetrically at 500 nm. Cholesterol concentration
is then calculated against a standard curve. The entire
assay is automated using a Biomek Automated Workstation.
Uterine Eosinophil Peroxidase (EPO) Assay
The uteri from above are kept at 4oC until time of
enzymatic analysis. The uteri are then homogenized in 50
volumes of 50 mM Tris buffer (pH - 8.0) containing 0.005
Triton X-100. Upon addition of 0.01 hydrogen peroxide and
10 mM O-phenylenediamine (final concentrations) in Tris
buffer, increase in absorbance is monitored for one minute
at 450 nm. The presence of eosonophils in the uterus is an
indication of estrogenic activity of a compound. The
maximal velocity of a 15 second interval is determined over
the initial, linear portion of the reaction curve.
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Inhibition of Bone Loss (Osteoporosis) Test Procedure
Following the general preparation procedure described
above, the rats are treated daily for thirty-five days (6
rats per treatment group) and sacrificed by carbon dioxide
asphyxiation on the 36th day. The thirty-five day time
period is sufficient to allow maximal reduction in bone
density, measured as described herein. At the time of
sacrifice, the uteri are removed, dissected free of
extraneous tissue, and the fluid contents are expelled
before determination of wet weight in order to confirm
estrogen deficiency associated with complete ovariectomy.
Uterine weight is routinely reduced about 75~ in response to
ovariectomy. The uteri are then placed in 10~ neutral
buffered formalin to allow for subsequent histological
analysis.
The right femurs are excised and digitilized X-rays
generated and analyzed by an image analysis program (NIH
image) at the distal metaphysis. The proximal aspect of the
tibiae from these animals are also scanned by quantitative
computed tomography. In accordance with the above
procedures, F-I or ethynyl estradiol (EE2) in 20~
hydroxypropyl ~-cyclodextrin are orally administered to test
animals. F-I is also useful in combination with estrogen or
progestin.
MCF-7 Proliferation Assay
MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are
maintained in MEM (minimal essential medium, phenol red-
free, Sigma, St. Louis, MO) supplemented with 10~ fetal
bovine serum (FBS) (V/V), L-glutamine (2 mM), sodium
pyruvate (1 mM), HEPES {(N-[2-hydroxyethyl]piperazine-N'-[2-
ethanesulfonic acid]10 mM}, non-essential amino acids and
bovine insulin (1 ug/mL) (maintenance medium). Ten days
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prior to assay, MCF-7 cells are switched to maintenance
medium supplemented with 10~ dextran coated charcoal
stripped fetal bovine serum (DCC-FBS) assay medium) in place
of 10~ FBS to deplete internal stores of steroids. MCF-7
cells are removed from maintenance flasks using cell
dissociation medium (Ca++/Mg++ free HBSS (phenol red-free)
supplemented with 10 mM HEPES and 2 mM EDTA). Cells are
washed twice with assay medium and adjusted to 80,000
cells/mL. Approximately 100 mL (8,000 cells) are added to
flat-bottom microculture wells (Costar 3596) and incubated
at 37oC in a 5~ C02 humidified incubator for 48 hours to
allow for cell adherence and equilibration after transfer.
Serial dilutions of drugs or DMSO as a diluent control are
prepared in assay medium and 50 mL transferred to triplicate
microcultures followed by 50 mL assay medium for a final
volume of 200 mL. After an additional 48 hours at 37oC in a
5~ C02 humidified incubator, microcultures are pulsed with
tritiated thymidine (1 uCi/well) for 4 hours. Cultures are
terminated by freezing at -70oC for 24 hours followed by
thawing and harvesting of microcultures using a Skatron
Semiautomatic Cell Harvester. Samples are counted by liquid
scintillation using a Wallac BetaPlace ~i counter.
DMBA-Induced Mammary Tumor Inhibition
Estrogen-dependent mammary tumors are produced in
female Sprague-Dawley rats which are purchased from Harlan
Industries, Indianapolis, Indiana. At about 55 days of age,
the rats receive a single oral feeding of 20 mg of 7,12-
dimethylbenz[a]anthracene (DMBA). About 6 weeks after DMBA
administration, the mammary glands are palpated at weekly
intervals for the appearance of tumors. V~henever one or
more tumors appear, the longest and shortest diameters of
each tumor are measured with a metric caliper, the
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measurements are recorded, and that animal is selected for
experimentation. An attempt is made to uniformly distribute
the various sizes of tumors in the treated and control
groups such that average-sized tumors are equivalently
distributed between test groups. Control groups and test
groups for each experiment contain 5 to 9 animals.
F-I is administered either through intraperitoneal
injections in 2~ acacia, or orally. Orally administered
compounds are either dissolved or suspended in 0.2 mL corn
oil. Each treatment, including acacia and corn oil control
treatments, is administered once daily to each test animal.
Following the initial tumor measurement and selection of
test animals, tumors are measured each week by the above-
mentioned method. The treatment and measurements of animals
continue for 3 to 5 weeks at which time the final areas of
the tumors are determined. For each compound and control
treatment, the change in the mean tumor area is determined.
Uterine Fibrosis Test Procedures
Test 1: Between 3 and 20 women having uterine fibrosis are
administered F-I. The amount of compound administered is
from 0.1 to 1000 mg/day, and the period of administration is
3 months. The women are observed during the period of
administration, and up to 3 months after discontinuance of
administration, for effects on uterine fibrosis.
Test 2: The same procedure is used as in Test 1, except the
period of administration is 6 months.
Test 3: The same procedure is used as in, Test 1, except the
period of administration is 1 year.
Test 4: Prolonged estrogen stimulation is used to induce
leiomyomata in sexually mature female guinea pigs. Animals
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are dosed with estradiol 3-5 times per week by injection for
2-4 months or until tumors arise. Treatment consisting of
F-I or vehicle is administered daily for 3-16 weeks and then
animals are sacrificed and the uteri harvested and analyzed
for tumor regression.
Test 5: Tissue from human leiomyomas are implanted into the
peritoneal cavity and/or uterine myometrium of sexually
mature, castrated, female, nude mice. Exogenous estrogen is
supplied to induce growth of the explanted tissue. In some
cases, the harvested tumor cells are cultured in vitro prior
to implantation. Treatment consisting of F-I or vehicle is
supplied by gastric lavage on a daily basis for 3-16 weeks
and implants are removed and measured for growth or
regression. At the time of sacrifice, the uteri are
harvested to assess the status of the organ.
Test 6: Tissue from human uterine fibroid tumors is
harvested and maintained, in vitro, as primary non-
transformed cultures. Surgical specimens are pushed through
a sterile mesh or sieve, or alternately teased apart from
surrounding tissue to produce a single cell suspension.
Cells are maintained in media containing 10~ serum and
antibiotic. Rates of growth in the presence and absence of
estrogen are determined. Cells are assayed for their
ability to produce complement component C3 and their
response to growth factors and growth hormone. In vitro
cultures are assessed for their proliferative response
following treatment with progestins, GnRH, F-I, and vehicle.
Levels of steroid hormone receptors are assessed weekly to
determine whether important cell characteristics are
maintained in vitro. Tissue from 5-25 patients is utilized.
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Test 7: F-I's ability to inhibit estrogen-stimulated
proliferation of leiomyoma-derived ELT cell lines is
measured substantially as described in Fuchs-Young, et al.,
"Inhibition of Estrogen-Stimulated Growth of Uterine
Leiomyomas by Selective Estrogen Receptor Modulators", Mol.
Car., 17(3):151-159 (1996), the teachings of which are
herein incorporated by reference.
Endometriosis Test Procedures
Test 1: Twelve to thirty adult CD strain female rats are
used as test animals. They are divided into three groups of
equal numbers. The estrous cycle of all animals is
monitored. On the day of proestrus, surgery is performed on
each female. Females in each group have the left uterine
horn removed, sectioned into small squares, and the squares
are loosely sutured at various sites adjacent to the
mesenteric blood flow. In addition, females in Group 2 have
the ovaries removed. On the day following surgery, animals
in Groups 1 and 2 receive intraperitoneal injections of
water for 14 days whereas animals in Group 3 receive
intraperitoneal injections of 1.0 mg of F-I per kilogram of
body weight for the same duration. Following 14 days of
treatment, each female is sacrificed and the endometrial
explants, adrenals, remaining uterus, and ovaries, where
applicable, are removed and prepared for histological
examination. The ovaries and adrenals are weighed.
Test 2: Twelve to thirty adult CD strain female rats are
used as test animals. They are divided into two equal
groups. The estrous cycle of all animals is monitored. On
the day of proestrus, surgery is performed on each female.
Females in each group have the left uterine horn removed,
sectioned into small squares, and the squares are loosely
sutured at various sites adjacent to the mesenteric blood
CA 02314682 2000-07-28
. ~. X-11357 24
flow. Approximately 50 days following surgery, animals
assigned to Group 1 receive intraperitoneal injections of
water for 21 days whereas animals in Group 2 receive
intraperitoneal injections of 1.0 mg of F-I per kilogram of
body weight for the same duration. Following 21 days of
treatment, each female is sacrificed and the endometrial
explants and adrenals are removed and weighed. The explants
are measured as an indication of growth. Estrous cycles are
monitored.
Test 3: Autographs of endometrial tissue are used to induce
endometriosis in rats and/or rabbits. Female animals at
reproductive maturity undergo bilateral oophorectomy, and
estrogen is supplied exogenously thus providing a specific
and constant level of hormone. Autologous endometrial
tissue is implanted in the peritoneum of 5-150 animals and
estrogen supplied to induce growth of the explanted tissue.
Treatment consisting of a compound of the present invention
is supplied by gastric lavage on a daily basis for 3-16
weeks, and implants are removed and measured for growth or
regression. At the time of sacrifice, the intact horn of
the uterus is harvested to assess status of endometrium.
Test 4: Tissue from human endometrial lesions is implanted
into the peritoneum of sexually mature, castrated, female,
nude mice. Exogenous estrogen is supplied to induce growth
of the explanted tissue. In some cases, the harvested
endometrial cells are cultured in vitro prior to
implantation. Treatment consisting of F-I supplied by
gastric lavage on a daily basis for 3-16 weeks, and implants
are removed and measured for growth or regression. At the
time of sacrifice, the uteri are harvested to assess the
status of the intact endometrium.
CA 02314682 2000-07-28
X-11357 25
Test 5: Tissue from human endometrial lesions is harvested
and maintained in vitro as primary non-transformed cultures.
Surgical specimens are pushed through a sterile mesh or
sieve, or alternately teased apart from surrounding tissue
to produce a single cell suspension. Cells are maintained
in media containing 10~ serum and antibiotic. Rates of
growth in the presence and absence of estrogen are
determined. Cells are assayed for their ability to produce
complement component C3 and their response to growth factors
and growth hormone. In vitro cultures are assessed for
their proliferative response following treatment with
progestins, GnRH, F-I, and vehicle. Levels of steroid
hormone receptors are assessed weekly to determine whether
important cell characteristics are maintained in vitro.
Tissue from 5-25 patients is utilized.
CNS Disorders Including Alzheimer's Disease
Estrogens, such as 17~-estradiol, regulate gene
transcription by binding to estrogen receptors (ER) which
reside in the cytoplasm of certain cell populations. Ligand
activation of the ER is a prerequisite for nuclear transport
of the complex where binding to a 13 base-pair palindromic
DNA consensus sequence (estrogen response element, or ERE)
begins assembly of a transcriptional apparatus which
culminates in the activation of appropriate target genes. A
variety of genes have been identified which are regulated by
estrogen. These include cytoskeletal proteins, neuro-
transmitter biosynthetic and metabolic enzymes and
receptors, as well as other hormones and neuropeptides.
ERE's have been identified in many estrogen-responsive genes
including vitellogenin, c-fos, prolactin, and luteinizing
hormone.
Of significance in the central nervous system, ERE-like
sequences have been identified in p75ngr and trkA, both of
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which serve as signaling molecules for the neurotrophins:
nerve growth factor (NGF), brain derived nerve growth factor
(BDNGF), and neurotrophin-3.
BDNF as well as NGF have been shown to promote the
survival of cholinergic neurons in culture. It is
postulated that if the interactions between neurotrophins
and estrogens are important for the development and survival
of basal forebrain neurons (which degenerate in Alzheimer's
disease) then clinical-conditions in which an estrogen
deficiency exists (as after menopause) may contribute to a
loss of these neurons.
The following experiment is conducted in ovariectomized
rats (prepared as described above) to determine the
similarities and/or differences between F-I and estrogen at
affecting gene expression in various brain regions. Six
week old rats are dosed daily with subcutaneous injections
of estradiol benzoate (0.03 mg/kg), F-I or vehicle
(control). After five weeks of treatment, animals are
sacrificed and their brains removed and hippocampi collected
by microdissection. The hippocampi are fast frozen in
liquid nitrogen and stored at -70oC. Total RNA is prepared
from pooled tissue from the appropriate treatment and
control groups and reverse transcribed using a 3'
oligonucleotide primer which is selected for specific mRNA
(poly-A+) populations. Polymerase chain reactions (PCR) are
carried out in a cocktail consisting of: random 5'
oligonucleotides (10 base-pairs in length; total of 150),
reaction buffer, Taq polymerase, and a 32PdTCP.
After 40 rounds of amplification, the reaction products
are size fractionated on a 6~ TBE-urea gel, dried and
exposed to X-ray film. The resulting mRNA display patterns
are compared between treatment groups.
CA 02314682 2000-07-28
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Use of F-I in Conjunction with Estrogen
Peri- and post-menopausal women often undergo hormone
replacement therapy (HRT) to combat negative consequences
associated with the drop in circulating endogenous estrogen,
e.g., to treat hot flashes. However, HRT has been
associated with increased risks of certain cancers including
uterine and breast cancer. F-I may be employed in
conjunction with HRT to inhibit these risks.
Use of F-I in Conjunction With an Aromatase Inhibitor
By definition, the ovaries of a postmenopausal woman
are not functioning. Her only source of estrogen is through
conversion of adrenal androgens to estrogens by the enzyme
aromatase, which is found in peripheral tissues (including
fat, muscle and the breast tumor itself). Thus, drugs that
inhibit aromatase (aromatase inhibitors) deplete the
postmenopausal woman of circulating estrogen. Estrogen
deprivation by means of aromatase inhibition is an important
treatment option for patients with metastatic breast cancer.
During therapy with an aromatase inhibitor, lack of
circulating estrogen may cause negative, unintended side-
effects, for example on serum lipid levels. F-I may be
employed to inhibit these negative effects.
Use of F-I in Conjunction with a LHRH Analogue
Continuous exposure to a LHRH (lutenizing hormone
releasing hormone) analogue inhibits estrogen production in
the premenopausal women by desensitizing the pituitary
gland, which then no longer stimulates the ovaries to
produce estrogen. The clinical effect is a "medical
oophrectomy" which is reversible upon cessation of the LHRH
analogue. During therapy with a LHRH analogue, lack of
circulating estrogen may cause negative, unintended side-
effects, for example on serum lipid levels. F-I may be
employed to inhibit these negative effects.
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Increasing Levels of Acetyl Choline
It is known that patients suffering from Alzheimer's
disease have a markedly smaller level of cholinergic neurons
in the hippocampus than their non-Alzheimer peers. The
progressive loss of these cholinergic neurons appears to
mirror the progressive loss in memory and cognitive function
in these patients. It is thought that one reason for the
decline of these neurons is the loss or decreased function
of the neurotransmitter, acetyl choline.
The level of acetylcholine in a neuron is basically
determined by where the equilibrium between its bio-
synthesis and bio-degradation lies. The enzyme choline
acetyltransferase (ChAT) is primarily responsible for its
synthesis and acetylcholineesterase (AChE) for its
degradation.
In the order to determine F-I's effect on levels of
ChAT, the following experiment is performed: Following the
general rat preparation procedure described above, 40 rats
are dosed daily by subcutaneous injection or oral gavage
with F-I at 3 mg/kg/day in a vehicle containing 10~
cyclodextrin, estradiol benzoate at 0.03 or 0.3 mg/kg/day,
or vehicle control. Animals are treated for 3 or 10 days.
There are twenty animals per each dosing regimen. At the
appropriate time intervals, the animals are sacrificed and
their brains dissected. The particular portions of the
brains are homogenized and assayed. Homogenates from the
hippocampus and frontal cortex were processed and
determination of ChAT activity is made by a radio-labelled
assay of the bio-synthesis of acetyl choline. This
procedure may be found in Schoepp et al., J. Neural
Transmiss., 78:183-193, 1989, the teachings of which are
incorporated by reference.
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~. X-11357 29
As expected, in the OVX animals, ChAT levels are
reduced >50~ (p<0.001) compared to the sham operated
controls.
In another embodiment of the present invention, F-I is
used in combination with an AChE inhibitor. Use of an AChE
inhibitor increases levels of acetylcholine by blocking its
degradation via inhibition of AChE.
Benign Prostatic Hyperplasia (BPH)
For background on the link between estrogen action and
treatment of BPH and prostate carcinoma, see PCT Application
No. WO 98/07274, International Publication Date: October 15,
1998.
In the experiments described below, the ability of F-I
to bind at estrogen receptors in several human prostatic
cancer cell lines is evaluated.
Lysates of the LNCaP, DU-45 and PC-3 human prostatic
cancer cell lines are prepared in a TEG medium comprising 50
nM Tris~HC1 pH 7.4, 1.5 mM ethylenediamine tetraacetic acid
(EDTA) 0.4 M KC1, 10~ glycerol, 0.5 mM 2-ME, and 10 mM
sodium molybdate further containing the protease inhibitors
pepstatin (1 mg/mL), leupeptin (2 mg/mL), aprotinin (5
mg/mL) and phenylmethylsulfonyl fluoride (PMSF, 0.1 mM)
( TEGP ) .
The cell lysates are centrifuged and the pellets
resuspended in cold TEGP (1 mL TEGP/100 mg of pellet) and
sonicated for 30 seconds (duty cycle 70$, output 1.8) on a
Branson Model 450 Sonifier. Lysates are pelleted by
centrifugation at 10,000 x G for 15 minutes at 4oC after
which the supernates are withdrawn and either used
immediately or stored at -70oC.
Competitive Binding Assay: The binding buffer is TEG in
which the 0.4 M KC1 is replaced by 50 mM NaCl and to which 1
mg/mL of ovalbumin had been further added (TEGO). F-I is
CA 02314682 2000-07-28
X-11357 30
diluted to 20 nM in TEGO from which 3-fold serial dilutions
are prepared. Assays are performed in round-bottom
polyprolylene microplates in triplicate microwells. Each
well receives 35 mL of tritiated 17(3-estradiol (0.5 nM,
specific activity 60.1 Ci/mmol, DuPont-New England Nuclear,
Boston, MA) and 35 mL of cold competitot test compound (0.1
nM - 5 mM) or TEGO, and following incubation for 5 minutes
at 4oC with shaking, 70 mL of MCF-7 cell line lysate.
Plates are incubated for 24 hours at 4oC after which
time 70 mL of dextran-coated charcoal (DCC) is added to each
well followed by vigorous shaking for 8 minutes at 4oC. The
plates are then centrifuged at 1500 x G for 10 minutes at
4oC. Supernate is harvested from each well into a flexible
polystyrene microplate for scintillation counting in a
lnlallac Micobeta Model 1450 counter. Radioactivity is
expressed as disintegrations per minute (DPM) after
correcting for counting efficiency (35-40~) and background.
Additional controls are total counts and total counts + DCC
to defined the lower limit of DCC extractable counts. The
results of these competitive binding assays are expressed as
mean percent bound (~ Bound) +/- standard deviation using
the formula:
DPMtest compound - DPMtotal count + DCC
Bound = x 100
DPMno test compound- DPMtotal count + DCC
Prevention of Breast Cancer
This invention also relates to the administration of F-
I to a recipient who is at risk of developing de novo breast
cancer. The term "de novo", as used herein, means the lack
of transformation or metamorphosis of normal breast cells to
cancerous or malignant cells in the first instance. Such a
transformation may occur in stages in the same or daughter
CA 02314682 2000-07-28
X-11357 31
cells via an evolutionary process or may occur in a single,
pivotal event. This de novo process is in contrast to the
metastasis, colonization, or spreading of already
transformed or malignant cells from the primary tumor site
to new locations.
A person who is at no particular risk of developing
breast cancer is one who may develop de novo breast cancer,
has no evidence or suspicion of the potential of the disease
above normal risk, and who has never had a diagnosis of
having the disease. The greatest risk factor contributing
to the development of breast carcinoma is a personal history
of suffering from the disease, or an earlier occurrence of
the disease, even if it is in remission with no evidence of
its presence. Another risk factor is family history of the
disease.
Induction of mammary tumors in rats by administration
of the carcinogen N-nitroso-N-methylurea is a well-accepted
animal model for the study of breast cancer and has been
found suitable for analyzing the effect of chemopreventive
agents.
In two separate studies, 55-day old female Sprague-
Dawley rats are given an intravenous (Study 1) or
intraperitoneal (Study 2) dose of 50 mg of N-nitroso-N-
methylurea per kilogram of body weight one week prior to
feeding ad libitum a diet into which varying amounts of F-I,
(Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-
dimethylethanamine base (tamoxifen base), or control are
blended.
In Study 1, the dietary doses of 60 mg/kg of diet and
20 mg/kg of diet translates into roughly comparable doses of
3 and 1 mg/kg of body weight for the test animals.
In Study 2, the dietary doses of 20, 6, 2, and 0.6
mg/kg of diet translates roughly into comparable doses of 1,
0.3, 0.1 and 0.03 mg/kg of body weight for the test animals.
CA 02314682 2000-07-28
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Rats are observed for evidence of toxicity and are
weighed and palpated for tumor formation once a week. The
animals are sacrificed after thirteen weeks (Study 1~ or
eighteen weeks (Study 2) and tumors are confirmed and
weighed at autopsy.
Formulations
The term "pharmaceutical" when used herein as an
adjective means substantially non-deleterious to the
recipient mammal. By "pharmaceutical formulation" it is
meant the carrier, diluent, excipients and active
ingredients) must be compatible with the other ingredients
of the formulation, and not deleterious to the recipient
thereof.
F-I is preferably formulated prior to administration.
The selection of the formulation should be decided by the
attending physician taking into considerations the same
factors involved with determining the effective amount.
The total active ingredients in such formulations
comprises from 0.1~ to 99.9 by weight of the formulation.
Preferably, no more than two active ingredients are
contained in said formulation. That is, it is preferred to
formulate F-I with a second active ingredient selected from
an estrogen, progestin, aromatase inhibitor, LHRH analogue
and AChE inhibitor. Most preferred formulations are those
where F-I is the sole active ingredient.
Pharmaceutical formulations of the present invention
are prepared by procedures known in the art using well known
and readily available ingredients. For example, F-I, either
alone, or in combination with an estrogen, progestin,
aromatase inhibitor, LHRH analogue or an AChE inhibitor
compound, are formulated with common excipients, diluents,
or carriers, and formed into tablets, capsules, suspensions,
solutions, injectables, aerosols, powders, and the like.
CA 02314682 2000-07-28
. ,, X-11357 33
Pharmaceutical compositions of this invention for
parenteral administration comprise sterile aqueous or non-
aqueous solutions, dispersions, suspensions, or emulsions,
as well as sterile powders which are reconstituted
immediately prior to use into sterile solutions or
suspensions. Examples of suitable sterile aqueous and non-
aqueous carriers, diluents, solvents or vehicles include
water, physiological saline solution, ethanol, polyols (such
as glycerol, propylene glycol, polyethylene glycol), and
the like), and suitable mixtures thereof, vegetable oils
(such as olive oil), and injectable organic esters such as
ethyl oleate. Proper fluidity is maintained, for example,
by the use of coating materials such as lecithin, by the
maintenance of proper particle size in the case of
dispersions and suspensions, and by the use of surfactants.
Parenteral compositions may also contain adjuvants such
as preservatives, wetting agents, emulsifying agents, and
dispersing agents. Prevention of the action of
microorganisms is ensured by the inclusion of antibacterial
and antifungal agents, for example, paraben, chlorobutanol,
phenol sorbic acid, and the like. It may also be desirable
to include isotonic agents such as sugars, sodium chloride,
and the like. Prolonged absorption of injectable
formulations may be brought about by the inclusion of agents
which delay absorption such as aluminum monostearate and
gelatin.
In some cases, in order to prolong the effect of the
drug, it is desirable to slow the absorption of the drug
following subcutaneous or intramuscular injection. This may
be accomplished by the use of a liquid suspension of
crystalline material of low water solubility or by
dissolving or suspending the drug in an oil vehicle. In the
case of the subcutaneous or intramuscular injection of a
suspension containing a form of the drug with low water
CA 02314682 2000-07-28
~. X-11357 34
solubility, the rate of absorption of the drug depends upon
its rate of dissolution.
Injectable "depot" formulations of F-I are made by
forming microencapsulated matrices of the drug in
biodegradable polymers such as poly(lactic acid),
poly(glycolic acid), copolymers of lactic and glycolic acid,
poly (orthoesters), and poly (anhydrides) these materials
which are described in the art. Depending upon the ratio of
drug to polymer and the characteristics of the particular
polymer employed, the rate of drug release can be
controlled.
Injectable formulations are sterilized, for example, by
filtration through bacterial-retaining filters, or by
presterilization of the components of the mixture prior to
their admixture, either at the time of manufacture or just
prior to administration (as in the example of a dual chamber
syringe package).
Solid dosage forms for oral administration include
capsules, tablets, pills, powders, and granules. In such
solid dosage forms, F-I is mixed with at least one inert,
pharmaceutical carrier such as sodium citrate, or dicalcium
phosphate, and/or (a) fillers or extenders such as starches,
sugars including lactose and glucose, mannitol, and silicic
acid, (b) binding agents such as carboxymethyl-cellulose and
other cellulose derivatives, alginates, gelatin,
poly(vinylpyrrolidine), sucrose and acacia, (c) humectants
such as glycerol, (d) disintegrating agents such as agar-
agar, calcium carbonate, sodium bicarbonate, potato or
tapioca starch, alginic acid, silicates and sodium
carbonate, (e) moisturizing agents such as glycerol; (f)
solution retarding agents such as paraffin, (g) absorption
accelerating agents such as quaternary ammonium compounds,
(h) wetting agents such as cetyl alcohol and glycerin
monostearate, (i) absorbents such as kaolin and bentonite
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clay, and (j) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols), sodium
lauryl sulfate, and mixtures thereof. In the case of
capsules, tablets and pills, the dosage form may also
contain buffering agents.
Solid compositions of a similar type may also comprise
the fill in soft or hard gelatin capsules using excipients
such as lactose as well as high molecular weight
polyethylene glycols) and the like.
Solid dosage forms such as tablets, dragees, capsules,
pills and granules can also be prepared with coatings or
shells such as enteric coatings or other coatings well known
in the pharmaceutical formulating art. The coatings may
contain opacifying agents or agents which release the active
ingredients) in a particular part of the digestive tract,
as for example, acid soluble coatings for release of the
active ingredients) in the stomach, or base soluble
coatings for release of the active ingredients) in the
intestinal tract.
The active ingredients) may also be microencapsulated
in a sustained-release coating, with the microcapsules being
made part of a pill of capsule formulation.
Liquid dosage forms for oral administration of F-I
include solution, emulsions, suspensions, syrups and
elixirs. In addition to the active components, liquid
formulations may include inert diluents commonly used in the
art such as water or other pharmaceutical solvents,
solubilizing agents and emulsifiers such as ethanol,
isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethyl formamide, oils (in particular, cottonseed, ground
nut, corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols), fatty
acid esters of sorbitol, and mixtures thereof.
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Besides inert diluents, the liquid oral formulations
may also include adjuvants such as wetting agents,
emulsifying and suspending agents, and sweetening,
flavoring, and perfuming agents.
Liquid suspension, in addition to the active
ingredients) may contain suspending agents such as
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol
and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite clay, agar-agar, and tragacanth,
and mixtures thereof.
Compositions for rectal or intravaginal administration
are prepared by mixing F-I with suitable non-irritating
excipients such as cocoa butter, polyethylene glycol or any
suppository wax which is a solid at room temperature, but
liquid at body temperature and therefore melt in the rectum
or vaginal cavity to release the active component(s). The
compounds are dissolved in the melted wax, formed into the
desired shape, and allowed to harden into the finished
suppository formulation.
F-I may also be administered in the form of liposomes.
As is know in the art, liposomes are generally derived from
phospholipids or other lipid substances. Lipososome
formulations are formed by mono- or multilamellar hydrated
liquid crystals which are dispersed in an aqueous medium.
Any non-toxic, pharmaceutical, and metabolizable lipid
capable of forming liposomes can be used. The present
compositions in liposome form can contain, in addition to F-
I, stabilizers, excipients, preservatives, and the like.
The preferred lipids are phospholipids and the phosphatidyl
cholines (lecithins), both natural and synthetic.
Methods for forming liposomes are know in the art as
described, for example, in Prescott, Ed., Methods in Cell
Biology, Volume XIV, Academic Press, New York, N. Y. (1976),
p. 33 et seq.
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The following formulation examples are illustrative
only and are not intended to limit the scope of the present
invention.
Formulation 1: Gelatin Capsules
Hard gelatin capsules are Quantity (mg/capsule)
prepared using the
following: Ingredient
F-I 0.1 - 1000
Starch, NF 0 - 650
Starch flowable powder 0 - 650
Silicone fluid 350 centistokes 0 - 15
The formulation above may be changed in compliance with the
reasonable variations provided.
A tablet formulation is prepared using the ingredients
below:
Formulation 2: Tablets
Ingredient Quantity (mg/tablet)
F-I 2.5 - 1000
Cellulose, microcrystalline 200 - 650
Silicon dioxide, fumed 10 - 650
Stearate acid 5 - 15
The components are blended and compressed to form tablets.
Formulation 3: Tablets containing approximately 10 and 50
mgs, respectively, of F-I may be prepared as follows:
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Ingredient Quantity Quantity
(mg/tablet) (mg/tablet)
F-I 11.3 56.5
Lactose Anhydrous 176.8 128.2
Lactose Spray Dried Special 44.2 32.0
Povidone 11.0 13.0
Polysorbate 80 2.5 2.6
Crosspovidone (Inside) 6.25 6.24
Crosspovidone (Outside) 6.25 6.5
Magnesium Stearate 1.5 1.7
Microcrystalline Cellulose 0.0 13.0
(Outside)
The components are blended and compressed to form tablets.
Alternatively, tablets each containing 2.5 - 1000 mg of
F-I are made up as follows:
Formulation 4: Tablets
Ingredient Quantity (mg/tablet)
F-I 25 - 1000
Starch 45
Cellulose, microcrystalline 35
Polyvinylpyrrolidone 4
(as 10~ solution in water)
Sodium carboxymethyl cellulose 4.5
Magnesium stearate 0.5
Talc 1
F-I, starch, and cellulose are passed through a No. 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 so produced are dried at 50o-60oC and passed
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through a No. 18 mesh U.S. sieve. The sodium carboxymethyl
starch, magnesium stearate, and talc, previously passed
through a No. 60 U.S. sieve, are then added to the granules
which, after mixing, are compressed on a tablet machine to
yield tablets.
Suspensions each containing 0.1 - 1000 mg of medicament
per 5 ml dose are made as follows:
Formulation 5: Suspensions
Ingredient Quantity (mg/5 ml)
F-I 0.1 - 1000 mg
Sodium carboxymethyl cellulose 50 mg
Syrup 1.25 mg
Benzoic acid solution 0.10 mL
Flavor q.v.
Color
.v.
Purified water to 5
The medicament 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 color are diluted with some of the water and added, with
stirring. Sufficient water is then added to produce the
required volume.
An aerosol solution is prepared containing the
following ingredients:
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Formulation 6: Aerosol
Ingredient Quantity (~ by
weight )
F-I 0.25
Ethanol 25.75
Propellant 22 (Chlorodifluoromethane) 70.00
F-I 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 fed to a
stainless steel container and diluted with the remaining
propellant. The valve units are then fitted to the
container.
Suppositories are prepared as follows:
Formulation 7: Suppositories
Ingredient Quantity (mg/suppository)
F-I 250
Saturated fatty acid 2,000
glycerides
F-I is passed through a No. 60 mesh U.S. sieve and suspended
in the saturated fatty acid glycerides previously melted
using the minimal necessary heat. The mixture is then
poured into a suppository mold of.nominal 2 g capacity and
allowed to cool.
An intravenous formulation is prepared as follows:
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Formulation 8: Intravenous Solution
Ingredient Quantity
F-I 25 mg
Isotonic saline 1,000 mL
The solution of the above ingredients is intravenously
administered to a patient at a rate of about 1 mL per
minute.
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Formulation 9: Combination Capsule I
Ingredient Quantity (mg/capsule)
F-I 50
Premarin 1
Avicel pH 101 50
Starch 1500 117.50
Silicon Oil 2
Tween 80 0.50
Cab-O-Sil 0.25
Formulation 10: Combination Capsule II
Ingredient Quantity (rng/capsule)
F-I 50
Norethylnodrel 5
Avicel pH 101 82.50
Starch 1500 gp
Silicon Oil 2
Tween 80 0.50
Formulation 11: Combination Tablet
Ingredient Quantity (mg/capsule)
F-I 50
Premarin 1
Corn Starch NF 50
Povidone, K29-32 6
Avicel pH 101 41.50
Avicel pH 102 136.50
Crospovidone XL10 2.50
Magnesium Stearate 0.50
Cab-O-Sil 0.50
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Dosage
The specific dose of F-I administered according to this
invention is determined by the particular circumstances
surrounding each situation. These circumstances include,
the route of administration, the prior medical history of
the recipient, the pathological condition or symptom being
treated, the severity of the condition/symptom being
treated, and the age and sex of the recipient.
Generally, an effective minimum daily dose of F-I is
about 1, 5, 10, 15, or 20 mg. Typically, an effective
maximum dose is about 800, 100, 60, 50, or 40 mg. Most
typically, the dose ranges between 15 mg and 60 mg. The
exact dose may be determined, in accordance with the
standard practice in the medical arts of "dose titrating"
the recipient; that is, initially administering a low dose
of the compound, and gradually increasing the does until the
desired therapeutic effect is observed.
Although it may be necessary to dose titrate the
recipient with respect to the combination therapies
discussed above, typical doses of active ingredients other
than F-III are as follows: ethynyl estrogen (0.01 - 0.03
mg/day), mestranol (0.05 - 0.15 mg/day), conjugated
estrogenic hormones (e.g., Premarin~, Wyeth-Ayerst; 0.3 -
2.5 mg/day), medroxyprogesterone (2.5 -10 mg/day),
norethylnodrel (1.0 - 10.0 mg/day), nonethindrone (0.5 - 2.0
mg/day), aminoglutemide (250-1250 mg/day, preferably 250 mg
four times per day), anastrazole (1-5 mg/day, preferably 1
mg once per day), letrozole (2.5-10 mg/day, preferably 2.5
mg once a day), formestane (250-1250 mg per week, preferably
250 mg twice weekly), exemestane (25-100 mg/day, preferably
25 mg once per day), goserlin (3-15 mg/three months,
preferably 3.6-7.2 mg once every three months) and
~
~~ X-11357
CA 02314682 2000-07-28
44
leuprolide (3-15 mg/month, preferably 3.75-7.5 mg once every
month).
Route of administration
F-I can be administered by a variety of routes
including oral, rectal, transdermal, subcutaneus,
intravenous, intramuscular, and intranasal. The method of
administration of each estrogen- and progestin-based agent
is consistent with that which is known in the art. F-I,
alone or in combination with estrogen, progestin, or an AChE
inhibitor generally will be administered in a convenient
formulation.
The pharmaceutical compositions of this invention may
be administered to humans and other mammals (e. g., dogs,
cats, horses, swine and the like) orally, rectally,
intravaginally, parenterally, topically, bucally or
sublingually, or nasally. The term "parenteral
administration" refers herein to modes of administration
which include intravenous, intramuscular, intraperitoneal,
instrasternal, subcutaneous, or intraarticular injection or
infusion.
Mode/Length of Administration
For the majority of the methods of the present
invention, F-I is administered continuously, from 1 to 3
times daily or as often as needed to deliver an effective
amount of F-I to the recipient. Cyclical therapy may
especially be useful in the treatment of endometriosis or
may be used acutely during painful attacks of the disease.
In the case of restenosis, therapy may be limited to short
(1-6 months) intervals following medical procedures such as
angioplasty.
