U.S. patent number RE38,968 [Application Number 10/375,339] was granted by the patent office on 2006-02-07 for methods for inhibiting bone loss using 6-hydroxy-2-(4-hydroxyphenyl)-benzo[b]thien-3-yl-4-[2-(piperidin-1-yl) ethoxyphenylimethanone hydrochloride.
This patent grant is currently assigned to Eli Lilly and Company, Eli Lilly and Company. Invention is credited to Larry J. Black, George J. Cullinan.
United States Patent |
RE38,968 |
Black , et al. |
February 7, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Methods for inhibiting bone loss using
6-hydroxy-2-(4-hydroxyphenyl)-benzo[b]thien-3-yl-4-[2-(piperidin-1-yl)
ethoxyphenylimethanone hydrochloride
Abstract
The current invention provides a method useful for inhibiting
the loss of bone using
6-hydroxy-2-(4-hydroxyphenyl)-benzo(B)-thien-3-yl-4[2-piperidin-1-ethoxyp-
henol]methanone hydrochloride.
Inventors: |
Black; Larry J. (Indianapolis,
IN), Cullinan; George J. (Trafalgar, IN) |
Assignee: |
Eli Lilly and Company
(Indianapolis, IN)
|
Family
ID: |
27391294 |
Appl.
No.: |
10/375,339 |
Filed: |
February 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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08180522 |
Jan 12, 1994 |
5393763 |
|
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07920933 |
Jul 28, 1992 |
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Reissue of: |
08329396 |
Oct 26, 1994 |
05457117 |
Oct 10, 1995 |
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Current U.S.
Class: |
514/337;
514/333 |
Current CPC
Class: |
A61K
31/40 (20130101); A61K 31/425 (20130101); A61K
31/445 (20130101); A61K 31/4535 (20130101); A61K
31/55 (20130101) |
Current International
Class: |
A61K
31/44 (20060101) |
Field of
Search: |
;514/333,337,443,448 |
References Cited
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Foreign Patent Documents
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0062505 |
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Oct 1982 |
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EP |
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0068563 |
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Jan 1983 |
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EP |
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068563 |
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Jan 1983 |
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EP |
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0605193 |
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Jul 1994 |
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EP |
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2097788 |
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Nov 1982 |
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GB |
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304924 |
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Dec 1992 |
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NO |
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WO93/10113 |
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May 1993 |
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WO |
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WO 93/10741 |
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Jun 1993 |
|
WO |
|
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|
Primary Examiner: Webman; Edward J.
Attorney, Agent or Firm: Woodard, Emhardt, Moriarity, McNett
& Henry LLP
Parent Case Text
This application is a division, of application Ser. No. 08/180,522
filed Jan. 12, 1994, now U.S. Pat. No. 5,393,763, which is a
continuation of application Ser. No. 07/920,933 filed Jul. 28,
1992, now abandoned.
Claims
We claim:
1. A method of inhibiting .Iadd.post-menopausal .Iaddend.bone loss
.Iadd.in a post-menopausal woman in need of treatment to prevent or
treat post-menopausal osteoporosis .Iaddend.comprising
administering to .[.a human.]. .Iadd.said woman .Iaddend.in need
.[.thereof.]. .Iadd.of said treatment .Iaddend.an effective amount
of
6-hydroxy-2-(4-hydroxyphenyl)-benzo[B]thien-3-yl-4-[2-(piperidin-1-yl)eth-
oxyphenyl]methanone hydrochloride.
.[.2. The method of claim 1 wherein said human is female..].
3. The method of claim .[.2.]. .Iadd.1 .Iaddend.wherein said
.[.human.]. .Iadd.woman .Iaddend.has .Iadd.post-menopausal
.Iaddend.osteoporosis.
.Iadd.4. A method of inhibiting post-menopausal bone loss in a
post-menopausal woman in need of treatment to prevent
post-menopausal osteoporosis comprising administering to said woman
in need of said treatment an effective amount of
6-hydroxy-2-(4-hydroxyphenyl)-benzo[b]thien-3-yl-4-[2-(piperidin-1-yl)eth-
oxyphenyl]methanone hydrochloride..Iaddend.
Description
BACKGROUND OF THE INVENTION
This invention relates to the discovery that a group of
2-phenyl-3-aroylbenzothiophenes is useful in the prevention of bone
loss.
The mechanism of bone loss is not well understood, but in practical
effect, the disorder arises from an imbalance in the formation of
new healthy bone and resorption of old bone, skewed toward a net
loss of bone tissue. This bone loss includes a decrease in both
mineral content and protein matrix components of the bone, and
leads to an increased fracture rate of, predominantly, femoral
bones and bones in the forearm and vertebrae. These fractures, in
turn, lead to an increase in general morbidity, a marked loss of
stature and mobility, and, in many cases, an increase in mortality
resulting from complications.
Bone loss occurs in a wide range of subjects, including
post-menopausal women, patients who have undergone hysterectomy,
patients who are undergoing or have undergone long-term
administration of corticosteroids, patients suffering from
Cushing's syndrome, and patients having gonadal .[.dysgensis.].
.Iadd.dysgenesis.Iaddend..
Unchecked, bone loss can lead to osteoporosis, a major debilitating
disease whose prominent feature is the loss of bone mass (decreased
density and enlargement of bone spaces) without a reduction in bone
volume, producing porosity and fragility.
One of the most common types of osteoporosis is found in
post-menopausal women affecting an estimated 20 to 25 million women
in the United States alone. A significant feature of
post-menopausal osteoporosis is the large and rapid loss of bone
mass due to the cessation of estrogen production by the ovaries.
Indeed, data clearly support the ability of estrogen to limit the
progression of osteoporotic bone loss, and estrogen replacement is
a recognized treatment for post-menopausal osteoporosis in the
United States and many other countries. However, although estrogens
have beneficial effects on bone, given even at very low levels,
long-term estrogen therapy has been implicated in a variety of
disorders, including an increase in the risk of uterine and breast
cancer, causing many women to avoid this treatment. Recently
suggested therapeutic regimens, which seek to lessen the cancer
risk, such as administering combinations of progestogen and
estrogen, cause the patient to experience regular withdrawal
bleeding, which is unacceptable to most older women. Concerns over
the significant undesirable effects associated with estrogen
therapy, and the limited ability of estrogens to reverse existing
bond loss, support the need to develop alternative therapy for bone
loss that generates the desirable effects on bone but does not
cause undesirable effects.
Attempts to fill this need by the use of compounds commonly known
as antiestrogens, which interact with the estrogen receptor, have
had limited success, perhaps due to the face that these compounds
generally display a mixed agonist/antagonist effect. That is,
although these compounds can antagonize estrogen interaction with
the receptor, the compounds themselves may cause estrogenic
responses is those tissues having estrogen receptors. Therefore,
some antiestrogens are subject to the same adverse effects
associated with estrogen therapy.
The current invention provides methods for inhibiting the loss of
bone without the associated adverse effects of estrogen therapy,
and thus serves as an effective and acceptable treatment for
osteoporosis.
The 2-phenyl-3-aroylbenzothiophene compounds that are the active
component in the formulations and methods of this invention were
first developed by C. David Jones and Tulio Suarez as
anti-fertility agents (see U.S. Pat. No. 4,133,814, issued Jan. 9,
1979). Certain compounds in the group were found to be useful in
suppressing the growth of mammary tumors.
Jones later found a group of related compounds to be useful for
antiestrogen and antiandrogen therapy, especially in the treatment
of mammary and prostatic tumors (see U.S. Pat. No. 4,418,068,
issued Nov. 29, 1983). One of these compounds, the compound of
formula I wherein X is a bond, R and R.sup.1 are hydroxyl, and
R.sup.2 is a piperidino ring, was clinically tested for a brief
time for the treatment of breast cancer. That compound is called
raloxifene, formerly keoxifene.
SUMMARY OF THE INVENTION
This invention provides new methods for the treatment of bone loss
comprising administering to a human in need of treatment an
effective amount of a compound of formula I ##STR00001## wherein X
is a bond, CH.sub.2, or CH.sub.2CH.sub.2; R and R.sup.1,
independently, are hydrogen, hydroxyl, C.sub.1-C.sub.6-alkoxy,
C.sub.1-C.sub.6-acyloxy,
C.sub.1-C.sub.6-alkoxy-C.sub.2-C.sub.6-acyloxy, R.sup.3-substituted
aryloxy, R.sup.3-substituted aroyloxy, R.sup.4-substituted
carbonyloxy, chloro, or bromo; R.sup.2 is a heterocyclic ring
selected from the group consisting of pyrrolidino, piperidino, or
hexamethyleneimino; R.sup.3 is C.sub.1-C.sub.3-alkyl,
C.sub.1-C.sub.3-alkoxy, hydrogen, or halo; and R.sup.4 is
C.sub.1-C.sub.6-alkoxy or aryloxy; or a pharmaceutically acceptable
salt thereof.
The invention also provides pharmaceutical formulations for
inhibiting bone loss comprising a compound of formula I, wherein R,
R1, R2, and X are as defined above in an amount that increases or
retains bone density, together with a pharmaceutically acceptable
carrier.
DETAILED DESCRIPTION OF THE INVENTION
The current invention concerns the discovery that a group of
2-phenyl-3-aroylbenzothiophenes (benzothiophenes) of formula I are
useful in the treatment of osteoporosis. The benzothiophenes of
formula I inhibit the loss of bone that results from a lack of
endogenous estrogen such as occurs in women following cessation of
menstruation due to natural, surgical, or other processes. The
reduction of bone density and mass that more rarely occurs in men
is also tied to the loss of hormonal regulation and is therefore
also a target for therapy according to the methods of the current
invention.
The benzothiophenes of formula I are a series of nonsteroidal
compounds that exhibit high affinity for conventional estrogen
receptors in primary sex target tissues. However, they elicit
minimal estrogenic responses in those tissues, and actually serve
as potent antagonists of natural estrogens such as estradiol. In
contrast to the report of Feldmann, S. et al., "Antiestrogen and
antiandrogen administration reduce bone mass in the rat", Bone and
Mineral, 7:245 (1989), the benzothiophenes of formula I are able to
antagonize classical estrogenic responses in primary sex target
tissues without significantly reducing bone density when given to
intact or estrogen treated animals, and they prevent bone loss in
estrogen deficient animals. This dichotomy indicates selective
agonist/antagonist actions on specific target cells which would
appear to be highly desirable in treatment of the menopausal
syndrome. Accordingly, the real benefit of the current discovery is
that the benzothiophenes of formula I inhibit the loss of bone but
do not elicit significant estrogenic responses in the primary sex
target tissues. Thus, the current invention provides a method of
inhibiting bone loss comprising administering to a human in need of
treatment an amount of a compound of formula I that inhibits bone
loss but does not significantly affect the primary sex target
tissues. This combination of features allows for long-term
treatment of the chronic ailment with a diminished risk of
developing the undesirable effects of customary estrogen
replacement therapy.
The biological action of the benzothiophenes of formula I is
complex and may be unrelated to the detectable presence of the
parent compound in the blood. Following oral administration of a
preferred benzothiophene of this invention, raloxifene (raloxifene
hydrochloride), to human subjects in the clinic, the parent
compound was not detected in the serum of those subjects. It was
determined that following oral administration, the compound was
extensively conjugated to the glucuronidated form and cleared
quickly from the bloodstream. Although no biological endpoints were
measured in the human recipients, there was concern that the
compound was not bioavailable.
Experiments were undertaken to address the bioavailability issue in
laboratory animals where biological activity could be assessed. The
animal studies indicated that raloxifene was maximally active in
inhibiting both uterine uptake of tritiated-estradiol and the
normal uterotrophic response to estradiol even under conditions
where raloxifene was extensively conjugated in the plasma of the
animals. Moreover, the conjugate, isolated from the urine of human
subjects treated with raloxifene, displayed significant
antiestrogenic/antiuterotrophic activity when administered
intravenously to rats, and inhibited the interaction of
tritiatedestradiol with rat uterine estrogen receptors in a manner
similar to the parent compound. These studies suggested the
conjugated compound may have been converted to the parental form at
the site of action, presumably by the action of
.beta.-glucuronidase. Such conversion may contribute to the
activity of the compound. .beta.-Glucuronidase is fairly ubiquitous
and is thought to be active in the resorption process of bone
remodeling, and would presumably be available for converting the
conjugated compound to the parental form if required for activity.
Therefore, conjugation of the benzothiophenes of formula I is not
considered to be necessarily detrimental to their bioavailability
as an inhibitor of bone loss.
Thus, the method of treatment provided by this invention is
practiced by administering to a human in need of inhibition of bone
loss, a dose of a compound of formula I or a pharmaceutically
acceptable salt thereof, that is effective to inhibit bone loss. A
particular benefit of this method is that it avoids potentially
harmful and unacceptable estrogenic side effects. The inhibition of
bone loss contemplated by the present method includes both medical
therapeutic and/or prophylactic treatment, as appropriate.
The method also includes the administration of a compound of
formula I given in combination with estrogen. The term estrogen as
used herein refers to any compound which approximates the spectrum
of activities of the naturally acting molecule which is commonly
believed to be 17.beta.-estradiol. Examples of such compounds
include estriol, estrone, ethynyl estradiol, Premarin (a commercial
preparation of conjugated estrogens isolated from natural
sources--Ayerst), and the like. Again, due to the selective
agonist/antagonist properties of the compounds of formula I, this
combination provides for the full benefits of estrogen therapy
without the concomitant adverse effects associated with estrogen
therapy alone.
The general chemical terms used in the description of a compound of
formula I have their usual meanings. For example, the term
"C.sub.1-C.sub.3-alkyl" includes such groups as methyl, ethyl,
propyl, and isopropyl.
The term "C.sub.1-C.sub.6-alkoxy" includes such groups as methoxy,
ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy and also includes
branched chain structures such as, for example, isopropoxy and
isobutoxy.
The term "C.sub.1-C.sub.6-acyloxy" includes methanoyloxy,
ethanoyloxy, propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy,
and the like and also includes branched chain structures such as,
for example, 2,2-dimethylpropanoyloxy, and
3,3-dimethylbutanoyloxy.
The term "C.sub.1-C.sub.6-alkoxy-C.sub.2-C.sub.6-acyloxy"
contemplates, for example, methoxyethanoyloxy, methoxypropanoyloxy,
methoxybutanoyloxy, methoxypentanoyloxy, methoxyhexanoyloxy,
ethoxyethanoyloxy, ethoxypropanoyloxy, ethoxybutanoyloxy,
ethoxypentanoyloxy, ethoxyhexanoyloxy, propoxyethanoyloxy,
propoxypropanoyloxy, propoxybutanoyloxy, and the like.
It should also be understood that as used herein, references to
alkyl and alkoxy structures also include cycloalkyl and cycloalkoxy
groups where the number of carbons within the structure is at least
3.
The terms "R.sup.3-substituted aryloxy" and "R.sup.3-substituted
aroyloxy" include such groups as phenyloxy, thienyloxy, furyloxy,
naphthyloxy, benzoyloxy, thienoyloxy, furoyloxy, naphthoyloxy, and
the like, where the R.sup.3 substitution group may be hydrogen,
hydroxyl, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy, or
halo.
The term "R.sup.4-substituted carbonyloxy, where the R.sup.4
substitution group may be C.sub.1-C.sub.6-alkoxy or aryloxy,
includes carbonate structures such as methoxycarbonyloxy
ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy,
pentyloxycarbonyloxy, hexyloxycarbonyloxy, phenyloxycarbonyloxy,
thienyloxycarbonyloxy, furyloxycarbonyloxy, and
naphthyloxycarbonyloxy.
Preferred methods of this invention comprise the use of compounds
of formula I wherein R and R.sup.1 are other than hydrogen, alkoxy,
aryloxy, chloro, or bromo and therefore represent ester and
carbonate configurations. Other preferred methods include the use
of formula I compounds wherein R and R.sup.1 are the same as one
another. Certain R.sup.2 groups also demonstrate preferable
characteristics when used in the methods of this invention. For
example, preferred methods of this invention include the use of
formula I compounds wherein R.sup.2 is piperidino or pyrrolidino,
especially piperidino. A further preferred subgroup of the
preferred piperidino and pyrrolidino compounds include compounds
wherein R and R.sup.1 are other than hydrogen and, in particular,
those wherein R and R.sup.1 are hydroxyl.
All of the compounds used in the methods of the current invention
can be made according to established procedures, such as those
detailed in U.S. Pat. Nos. 4,133,814 and 4,418,068. In general, the
process starts with a benzo[b]thiophene having a 6-hydroxyl group
and a 2-(4-hydroxyphenyl) group. The starting compound is
protected, alkylated, and deprotected to form the formula I
compounds wherein R and R.sup.1 are both hydroxy. The formula I
compounds that are ethers, esters, and carbonates may then be
formed if desired. Examples of the preparation of such compounds
are provided in the U.S. patents discussed above. Specific
preparations of yet other derivatized compounds useful in the
current invention are outlined in the Preparations sections below.
Modifications to the above methods may be necessary to accommodate
reactive functionalities of particular substituents. Such
modifications would be both apparent to, and readily ascertained
by, those skilled in the art.
The compounds used in the methods of this invention form
pharmaceutically acceptable acid and base addition salts with a
wide variety of organic and inorganic acids and bases and include
the physiologically acceptable salts which are often used in
pharmaceutical chemistry. Such salts are also part of this
invention. Typical inorganic acids used to form such salts include
hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,
phosphoric, hypophosphoric and the like. Salts derived from organic
acids, such as aliphatic mono and dicarboxylic acids, phenyl
substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic
acids, aromatic acids, aliphatic and aromatic sulfonic acids, may
also be used. Such pharmaceutically acceptable salts thus include
acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate,
benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, methylbenzoate, o-acetoxybenzoate,
naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,
.beta.-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate,
caprate, caprylate, chloride, cinnamate, citrate, formate,
fumarate, glycollate, heptanoate, hippurate, lactate, malate,
maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate,
isonicotinate, nitrate, oxalate, phthalate, terephthalate,
phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, propiolate, propionate,
phenylpropionate, salicylate, sebacate, succinate, suberate,
sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,
benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,
ethanesulfonate, 2-hydroxyethanesulfonate, methane-sulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate,
p-toluenesulfonate, xylenesulfonate, tartarate, and the like.
In addition, some of the formula I compounds may form solvates with
water or organic solvents such as ethanol. These solvates are also
contemplated for use in the methods of this invention.
The pharmaceutically acceptable acid addition salts are typically
formed by reacting a compound of formula I with an equimolar or
excess amount of acid. The reactants are generally combined in a
mutual solvent such as diethyl ether or benzene. The salt normally
precipitates out of solution within about one hour to 10 days and
can be isolated by filtration or the solvent can be stripped off by
conventional means.
Bases commonly used for formation of salts include ammonium
hydroxide and alkali and alkaline earth metal hydroxides,
carbonates and bicarbonates, as well as aliphatic and aromatic
amines, aliphatic diamines and hydroxy alkylamines. Bases
especially useful in the preparation of addition salts include
ammonium hydroxide, potassium carbonate, sodium bicarbonate,
calcium hydroxide, methylamine, diethylamine, ethylene diamine,
cyclohexylamine and ethanolamine.
The pharmaceutically acceptable salts generally have enhanced
solubility characteristics compared to the compound from which they
are derived, and thus are often more amenable to formulation as
liquids or emulsions.
The current invention also provides useful pharmaceutical
formulations for inhibiting bone loss comprising a formula I
compound plus one or more pharmaceutically acceptable excipients.
Pharmaceutical formulations can be prepared by procedures known in
the art. For example, the compounds can be formulated with common
excipients, diluents, or carriers, and formed into tablets,
capsules, suspensions, powders, and the like. Examples of
excipients, diluents, and carriers that are suitable for such
formulations include the following: fillers and extenders such as
starch, sugars, mannitol, and silicic derivatives; binding agents
such as carboxymethyl cellulose and other cellulose derivatives,
alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents
such as glycerol; disintegrating agents such as agaragar, calcium
carbonate, and sodium bicarbonate; agents for retarding dissolution
such as paraffin; resorption accelerators such as quaternary
ammonium compounds; surface active agents such as cetyl alcohol,
glycerol monostearate; adsorptive carriers such as kaolin and
bentonite; and lubricants such as talc, calcium and magnesium
stearate, and solid polyethyl glycols.
The compounds can also be formulated as elixirs or solutions for
convenient oral administration or as solutions appropriate for
parenteral administration, for instance by intramuscular,
subcutaneous or intravenous routes. Additionally, the compounds are
well suited to formulation as sustained release dosage forms and
the like. The formulations can be so constituted that they release
the active ingredient only or preferably in a particular part of
the intestinal tract, possibly over a period of time. The coatings,
envelopes, and protective matrices may be made, for example, from
polymeric substances or waxes.
The particular dosage of a compound of formula I required to treat
or inhibit bone loss according to this invention will depend upon
the severity of the disease, its route of administration, and
related factors that will be decided by the attending physician.
Generally, accepted and effective doses will be from about 0.1 to
about 1000 mg, and more typically from about 200 to about 600 mg.
Such dosages will be administered to a subject in need of treatment
from once to about three times each day, or more often as needed to
effectively inhibit the bone loss process.
It is usually preferred to administer a compound of formula I in
the form of an acid addition salt, as is customary in the
administration of pharmaceuticals bearing a basic group such as the
piperidino ring. It is also advantageous to administer such a
compound by the oral route to an aging human (e.g. a
post-menopausal female or a male showing evidence of bone loss by
X-ray analysis). For such purposes the following oral dosage forms
are available.
FORMULATIONS
In the formulations which follow, "Active ingredient" means a
compound of formula I.
TABLE-US-00001 Formulation 1: Gelatin Capsules Hard gelatin
capsules are prepared using the following: Ingredient Quantity
(mg/capsule) Active ingredient 0.1-1000 Starch, NF 0-650 Starch
flowable powder 0-650 Silicone fluid 350 centistokes 0-15
The ingredients are blended, passed through a No. 45 mesh U.S.
sieve, and filled into hard gelatin capsules.
Examples of specific capsule formulations containing raloxifene
that have been made include those shown below:
TABLE-US-00002 Formulation 2: Raloxifene capsule Ingredient
Quantity (mg/capsule) Raloxifene 1 Starch, NF 112 Starch flowable
powder 225.3 Silicone fluid 350 centistokes 1.7
TABLE-US-00003 Formulation 3: Raloxifene capsule Ingredient
Quantity (mg/capsule) Raloxifene 1 Starch, NF 112 Starch flowable
powder 225.3 Silicone fluid 350 centistokes 1.7
TABLE-US-00004 Formulation 4: Raloxifene capsule Ingredient
Quantity (mg/capsule) Raloxifene 10 Starch, NF 103 Starch flowable
powder 225.3 Silicone fluid 350 centistokes 1.7
TABLE-US-00005 Formulation 5: Raloxifene capsule Ingredient
Quantity (mg/capsule) Raloxifene 50 Starch, NF 150 Starch flowable
powder 397 Silicone fluid 350 centistokes 3.0
The specific formulations above may be changed in compliance with
the reasonable variations provided.
A tablet formulation is prepared using the ingredients below:
TABLE-US-00006 Formulation 6: Tablets Ingredient Quantity
(mg/tablet) Active ingredient 0.1-1000 Cellulose, microcrystalline
0-650 Silicon dioxide, fumed 0-650 Stearate acid 0-15
The components are blended and compressed to form tablets.
Alternatively, tablets each containing 0.1-1000 mg of active
ingredient are made up as follows:
TABLE-US-00007 Formulation 7: Tablets Ingredient Quantity
(mg/tablet) Active ingredient 0.1-1000 Starch 45 Cellulose,
microcrystalline 35 Polyvinylpyrrolidone 4 (as 10% solution in
water) Sodium carboxymethyl cellulose 4.5 Magnesium stearate 0.5
Talc 1
The active ingredient, 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 50.degree.-60.degree. C. and passed through a
No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium
stearate, and talc, previously passed through a No. 60 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:
TABLE-US-00008 Formulation 8: Suspensions Ingredient Quantity (mg/5
ml) Active ingredient 0.1-1000 mg Sodium carboxymethyl cellulose 50
mg Syrup 1.25 mg Benzoic acid solution 0.10 mL Flavor q.v. Color
q.v. Purified water to 5 mL
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.
Illustrative compounds that can be used in the formulations and
methods of this invention are shown in Table 1.
TABLE-US-00009 TABLE 1 Compound No. X R and R.sup.1 R.sup.2 Form 1
bond ##STR00002## piperidino base 2 bond ##STR00003## piperidino
HCl 3 bond ##STR00004## piperidino base 4 bond ##STR00005##
piperidino HCl 5 bond --OC(O)CH.sub.2CH.sub.2CH.sub.3 piperidino
base 6 bond --OC(O)CH.sub.2CH.sub.2CH.sub.3 piperidino HCl 7 bond
--OC(O)C(CH.sub.3).sub.3 piperidino base 8 bond
--OC(O)C(CH.sub.3).sub.3 piperidino HCl 9 bond
--OC(O)CH.sub.2C(CH.sub.3).sub.3 piperidino base 10 bond
--OC(O)CH.sub.2C(CH.sub.3).sub.3 piperidino HCl 11 bond
##STR00006## piperidino HCl 12 bond ##STR00007## piperidino base 13
bond --OC(O)OCH.sub.2CH.sub.2CH.sub.2CH.sub.3 piperidino base 14
bond --OC(O)OCH.sub.2CH.sub.2CH.sub.2CH.sub.3 piperidino HCl 15
bond ##STR00008## piperidino base 16 bond ##STR00009## piperidino
HCl 17 bond ##STR00010## piperidino base 18 bond
--OC(O)CH.sub.2CH.sub.2OCH.sub.3 piperidino base 19 bond
--OC(O)CH.sub.2CH.sub.2OCH.sub.3 piperidino HCl 20 bond OH
piperidino base 21 bond OH piperidino HCl 22 bond H piperidino base
23 CH.sub.2 OH piperidino HCl 24 CH.sub.2CH.sub.2 OH piperidino HCl
25 CH.sub.2 H piperidino HCl 26 bond OH pyrrolodino base 27 bond OH
pyrrolodino HCl 28 CH.sub.2 OH pyrrolodino HCl 29 CH.sub.2CH.sub.2
OH pyrrolodino HCl 30 bond H pyrrolodino HCl 31 bond OH
hexamethyleneimino HCl 32 CH.sub.2 OH hexamethyleneimino HCl 33
CH.sub.2CH.sub.2 OH hexamethyleneimino HCl 34 bond OCH.sub.2
piperidino HCl
In the following Preparations, the compound numbers correspond to
those given in Table 1
Preparation 1
Preparation of Compound 1:
6-(4-Fluorobenzoyloxy)-2-[4-(4-fluorobenzoyloxy)phenyl]benzo[b]thien-3-yl-
-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.
Raloxifene,
6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl-4-[2-(piperidin-1-yl)etho-
xyphenyl]methanone hydrochloride, (5.1 g, 10 mmol) was suspended in
250 mL of dry tetrahydrofuran (THF) and 7.1 g (70 mmol) of
triethylamine, and approximately 10 mg of
4-(N,N-dimethylamino)pyridine were added. The suspension was cooled
in an ice bath and placed under an atmosphere of nitrogen.
4-Fluorobenzoyl chloride (4.75 g, 30 mmol), dissolved in 20 mL of
dry THF, was slowly added over a twenty minute period. The reaction
mixture was stirred and allowed to slowly warm to room temperature
over a period of eighteen hours. It was then filtered, and the
filtrate was evaporated to a gum in vacuo. The crude product thus
obtained was dissolved in a small volume of chloroform and
chromotagraphed (HPLC) on a silica gel column eluted with a linear
gradient of solvent, starting with chloroform and ending with a
mixture of chloroform-methanol (19:1 (v/v)). The fractions
containing the desired product as determined by thin layer
chromatography (silica, chloroform-methanol (9:1)) were combined
and evaporated to a gum. The final product was crystallized from
ether to give 3.21 g of compound 1.
PMR: consistent with the structure FDMS: m/e=717 M+ Elemental
Analysis for C.sub.42H.sub.33F.sub.2NO.sub.6S: Theor: C, 70.29; H,
4.60; N, 1.95 Found: C, 70.05; H, 4.60; N, 1.89 Mol. Wt.: 717
Preparation 2
Preparation of Compound 2:
6-(4-Fluorobenzoyloxy)-2-[4-(4-fluorobenzoyloxy)phenyl]benzo[b]thien-3-yl-
-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride.
Compound 1 (5.15 g, 7.18 mmol) was dissolved in 25 mL THF, and 150
mL ether was added. Dry HCl gas was bubbled into the solution, and
a white gummy precipitate formed. The liquid was removed by
decanting, and the residue was crystallized from ethyl acetate with
a small amount of ethanol added to effect solution. The product was
filtered, washed with ether, and dried to give 4.41 g of Compound 2
as a white powder.
PMR: consistent with the structure Elemental Analysis for
C.sub.42H.sub.34l ClF.sub.2NO.sub.6S: Theor: C, 66.88; H, 4.54; N,
1.86 Found: C, 66.59, H, 4.39; N, 1.60 Mol. Wt.: 753.5
Preparation 3
Preparation of Compound 3:
6-(Cyclopropylcarbonyloxy)-2-[4-(cyclopropylcarbonyloxy)phenyl]benzo[b]th-
ien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.
The title compound was prepared using procedures analogous to those
in Preparation 1, but using cyclopropylcarbonyl chloride, except
that the product was not crystallized. Yield=2.27 g.
PMR: consistent with the structure FDMS: m/e=610 M.sup.+
Preparation 4
Preparation of Compound 4:
6-(Cyclopropylcarbonyloxy)-2-[4-(cyclopropylcarbonyloxy)phenyl]benzo[b]th-
ien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
hydrochloride.
Compound 4 was prepared from Compound 3 as described in Preparation
2.
Preparation 5
Preparation of Compound 5:
6-(n-Butanoyloxy)-2-[4-(n-butanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(pi-
peridin-1-yl)ethoxy]phenyl]methanone.
Compound 5 was prepared using the method of Preparation 1, but
starting with n-butanoyl chloride, to give 4.12 g of final product
as an oil.
PMR: consistent with the structure FDMS: m/e=614 (M.sup.+1)
Preparation 6
Preparation of Compound 6:
6-(n-Butanoyloxy)-2-[4-(n-butanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(pi-
peridin-1-yl)ethoxy]phenyl]methanone hydrochloride.
Compound 5 (4.12 g) was dissolved in ethyl acetate (50 mL), and a
solution of HCl in ether was added until the precipitation stopped.
The liquid was decanted off, and the white, gummy residue was
triturated with diethyl ether and filtered. The residue was dried
to give 1.33 g of Compound 6.
PMR: consistent with the structure Elemental Analysis of for
C.sub.36H.sub.40ClNO.sub.6S: Theor.: C, 66.50; H, 6.20; N, 2.15
Found: C, 66.30; H, 6.28; N, 1.98 Mol. Wt.: 650.24
Preparation 7
Preparation of Compound 7:
6-(2,2-Dimethylpropanoyloxy)-2-[4-(2,2-dimethylpropanoyloxy)phenyl]benzo[-
b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.
Compound 7 was prepared using the procedure of Preparation 1, but
using 2,2-dimethylpropanoyl chloride.
Preparation 8
Preparation of Compound 8:
6-(2,2-Dimethylpropanoyloxy)-2-[4-(2,2-dimethylpropanoyloxy)phenyl]benzo[-
b]thien-3-yl-[4-[2-(piperidin-1yl)ethoxy]phenyl]methanone
hydrochloride
Compound 8 was prepared from Compound 7, as described in
Preparation 2.
FDMS: m/e=641 (M-HCl-1) Elemental Analysis of
C.sub.38H.sub.44ClNO.sub.6S: Theor.: C, 67.2 9; H, 6.54; N, 2.07
Found: C, 67.02; H, 6.54; N, 1.90 Mol. Wt.: 678.29
Preparation 9
Preparation of Compound 9:
6-(3,3-Dimethylbutanoyloxy)-2-[4-(3,3-dimethylbutanoyloxy)phenyl]benzo[b]-
thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.
Compound 9 was prepared using the procedure of Preparation 1, but
with 3,3-dimethylbutanoyl chloride.
Preparation 10
Preparation of Compound 10:
6-(3,3-Dimethylbutanoyloxy)-2-[4-(3,3-dimethylbutanoyloxy)phenyl]benzo[b]-
thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
hydrochloride.
Compound 10 was prepared from Compound 9 as described in
Preparation 2.
FDMS: m/e=669 (M-HCl-1) Elemental Analysis of
C.sub.40H.sub.48ClNO.sub.6S: Theor.: C, 68.02; H, 6.85; N, 1.98
Found: C, 67.75; H, 6.83; N, 2.04 Mol. Wt.: 706.35
Preparation 11
Preparation of Compound 11:
6-(4-Methylbenzoyloxy)-2-[4-(4-methylbenzoyloxy)phenyl]benzo[b]thien-3-yl-
[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride.
Compound 11 was prepared from the free base using a procedure
similar to that of Preparation 2.
FDMS: m/e=710 (M-HCl-1) Elemental Analysis of
C.sub.44H.sub.40ClNO.sub.6S: Theor.: C, 70.81; H, 5.39; N, 1.88
Found: C, 71.10; H, 5.39; N, 1.94 Mol. Wt.: 746.33
Preparation 12
Preparation of Compound 12:
6-Benzoyloxy-2-[4-benzoyloxy)phenoyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-
-yl)ethoxy]-phenyl]methanone.
Compound 12 was prepared from the appropriate acid chloride as
described in Preparation 1.
FDMS: m/e=682 (M+1) Elemental Analysis of
C.sub.42H.sub.35NO.sub.6S: Calc: C, 73.80; H, 5.14; N, 2.05 Found:
C, 73.27; H, 5.27; N, 1.94 Mol. Wt.: 681.8
Preparation 13
Preparation of Compound 13:
6-(n-Butoxyoyloxy)-2-[4(n-butoxyoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(pi-
peridin-1-yl)ethoxy]phenyl]methanone.
Compound 13 was prepared in a manner analogous to that described in
Preparation 1, except that n-butylchloroformate was used in place
of the acid chloride. Yield=6.13 g in form of oil.
PMR: consistent with structure FDMS: m/e=674 (M+1)
Preparation 14
Preparation of Compound 14:
6-(n-Butoxycarbonyloxy)-2-[4(n-butoxycarbonyloxy)phenyl]benzo[b]thien-3-y-
l[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride.
Compound 13 was converted to the hydrochloride salt in a manner
analogous to that described in Preparation 6.
PMR: consistent with structure Elemental Analysis of
C.sub.38H.sub.44ClNO.sub.SS: Calc: C, 64.26; H, 6.24; N, 1.97
Found: C, 63.97; H, 6.34; N, 1.98 Mol. Wt.: 710.29
Preparation 15
Preparation of Compound 15:
6-(Phenyloxycarbonyloxy)-2-[4(phenyloxycarbonyloxy)phenyl]benzo[b]thien-3-
-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.
This compound was prepared in a manner analogous to that described
in Preparation 13, but using the appropriate acyl ester. Yield=3.59
g of final product as a tan amorphous powder.
PMR: consistent with structure FDMS: m/e=713 (M+).
Preparation 16
Preparation of Compound 16:
6-(Phenyloxycarbonyloxy)-2-[4(phenyloxycarbonyloxy)phenyl]benzo[b]thien-3-
-yl[4-[2-(piperidin-1-yl)ethoxy)phenyl]methanone hydrochloride.
Compound 15 was converted to the hydrochloride salt in a manner
analogous to that described in Preparation 6.
PMR: consistent with structure Elemental Analysis of
C.sub.38H.sub.44ClNO.sub.8S: Calc: C, 67.24; H, 4.84; N, 1.87
Found: C, 66.94; H, 4.96; N, 1.84 Mol. Wt.: 750.27
Preparation 17
Preparation of Compound 17:
6-(Naphthoyloxy)-2-[4(1-naphthoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(pipe-
ridin-1-yl)ethoxy]phenyl]methanone.
Compound 17 was prepared as described in Preparation 1 using the
appropriate acid halide. Yield=3.5 g of a white amorphous
powder
PMR: consistent with structure FDMS: m/e=781 (M+) Elemental
Analysis of C.sub.50H.sub.39NO.sub.6S: Calc: C, 76.80; H, 5.03; N,
1.79 Found: C, 76.53; H, 5.20; N, 1.53 Mol. Wt.: 781.94
Preparation 18
Preparation of Compound 18:
6-(Methoxyethanoyloxy)-2-[4(methoxyethanoyloxy)phenyl]benzo[b]thien-3-yl[-
4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.
Compound 18 was prepared as described in Preparation 1 using the
appropriate acid halide. Yield=3.61 g of a gummy solid.
PMR: consistent with structure FDMS: m/e=618 (M+1)
Preparation 19
Preparation of Compound 19:
6-(Methoxyethanoyloxy)-2-[4(methoxyethanoyloxy)phenyl]benzo[b]thien-3-yl[-
4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride.
Compound 19 was prepared from 3.5 g of Compound 18 as described in
Preparation 2. Yield=1.65 g of amorphous white powder.
PMR: consistent with structure FDMS: m/e=618 (M+1) Elemental
Analysis of C.sub.34H.sub.36NO.sub.8S: Calc: C, 62.43; H, 5.55; N,
2.14 Found: C, 62.23; H, 5.63; N, 2.15
The following nonlimiting examples illustrate the methods and
formulations of this invention.
EXAMPLE 1
In the examples illustrating the methods, a model of
post-menopausal osteoporosis was used in which effects of different
treatments upon femur density were determined.
Seventy-five day old female Sprague Dawley rats (weight range of
225 to 275 g) were obtained from Charles River Laboratories
(Portage, Mi.). They were housed in groups of 3 and had ad libitum
access to food (calcium content approximately 1%) and water. Room
temperature was maintained at 22.2.degree..+-.1.7.degree. C. with a
minimum relatively humidity of 40%. The photoperiod in the room was
12 hours light and 12 hours dark.
One week after arrival, the rats underwent bilateral ovariectomy
under anesthesia (44 mg/kg Ketamine and 5 mg/kg Xylazine (Butler,
Indianapolis, Ind.) administered intramuscularly). Treatment with
vehicle, estrogen, or a compound of formula I was initiated on the
day of surgery following recovery from anesthesia. Oral dosage was
by gavage in 0.5 mL of 1% carboxymethylcellulose (CMC). Body weight
was determined at the time of surgery and weekly thereafter and the
dosage was adjusted with changes in body weight. Vehicle or
estrogen treated ovariectomized (ovex) rats and non-ovariectomized
(intact) rats were evaluated in parallel with each experimental
group to serve as negative and positive controls.
The rats were treated daily for 35 days (6 rats per treatment
group) and sacrificed by decapitation on the 36th day. The 35 day
time period was sufficient to allow maximal reduction in bone
density, measured as described herein. At the time of sacrifice,
the uteri were removed, dissected free of extraneous tissue, and
the fluid contents were expelled before determination of wet weight
in order to confirm estrogen deficiency associated with complete
ovariectomy. Uterine weight was routinely reduced about 75% in
response to ovariectomy. The uteri were then placed in 10% neutral
buffered formalin to allow for subsequent histological
analysis.
The right femurs were excised and scanned at the distal metaphysis
1 mm from the patellar groove with single photon absorptiometry.
Results of the densitometer measurements represent a calculation of
bone density as a function of the bone mineral content and bone
width.
INFLUENCE OF RALOXIFENE ON BONE DENSITY
The results of control treatments from five separate experiments
are accumulated in Table 2. In summary, ovariectomy of the rats
caused a reduction in femur density of about 25% as compared to
intact vehicle treated controls. Estrogen, administered in the
orally active form of ethynyl estradiol (EE.sub.2), prevented this
loss of bone in a dose dependent manner, but it also exerted a
stimulatory action on the uterus resulting in uterine weights
approaching that of an intact rat when administered at 100
.mu.g/kg. Results are reported as the mean of measurements from
thirty rats.+-.the standard error of the mean.
In these studies, raloxifene also prevented bone loss in a dose
dependent manner; however, only minimal increase of uterine weight
over the ovariectomized controls was present in these animals. The
results of five assays using raloxifene are combined in Table 3.
Accordingly, each point reflects the responses of thirty rats and
depicts a typical dose response curve for raloxifene in this model.
Results are reported as the mean .+-. the standard error of the
mean.
TABLE-US-00010 TABLE 2 Bone Density Uterine Weight (mg/cm/cm) (mg)
Ovariectomy control 170 .+-. 3 127 .+-. 5 (0.5 mL CMC oral) Intact
control 220 .+-. 4 545 .+-. 19 (0.5 mL CMC oral) EE.sub.2 100
.mu.g/kg, oral 210 .+-. 4 490 .+-. 11
TABLE-US-00011 TABLE 3 Bone Density Uterine Weight (mg/cm/cm) (mg)
Ovariectomy control 171 .+-. 3 127 .+-. 5 (0.5 mL CMC oral) Intact
control 222 .+-. 3 540 .+-. 22 (0.5 mL CMC oral) raloxifene 0.01
mg/kg, oral 176 .+-. 3 150 .+-. 5 raloxifene 0.10 mg/kg, oral 197
.+-. 3 196 .+-. 5 raloxifene 1.00 mg/kg, oral 201 .+-. 3 199 .+-. 5
raloxifene 10.00 mg/kg, oral 199 .+-. 3 186 .+-. 4
EXAMPLE 2
Raloxifene was administered alone or in combination with ethynyl
estradiol. Rats treated with raloxifene alone had uterine weights
which were marginally higher than the ovariectomized controls and
much less than those of ethynyl estradiol treated rats, which
approached those of the intact controls. Conversely, raloxifene
treatment significantly reduced bone loss in ovariectomized rats,
and when given in combination with ethynyl estradiol it did not
appreciably reduce the protective effect of the estrogen on bone
density. The results are shown in Table 4.
TABLE-US-00012 TABLE 4 Bone Density Uterine Weight (mg/cm/cm) (mg)
Experiment A Ovariectomy control 162 .+-. 4 142 .+-. 18 (0.5 mL CMC
oral) Intact control 219 .+-. 5 532 .+-. 49 (0.5 mL CMC oral)
EE.sub.2 100 .mu.g/kg, oral 202 .+-. 6 450 .+-. 17 EE.sub.2 100
.mu.g/kg + 204 .+-. 2 315 .+-. 10 raloxifene 0.10 mg/kg, oral
EE.sub.2 100 .mu.g/kg + 200 .+-. 5 250 .+-. 21 raloxifene 1 mg/kg,
oral Experiment B Ovariectomy control 165 .+-. 8 116 .+-. 6 (0.5 mL
CMC oral) Intact control 220 .+-. 4 605 .+-. 69 (0.5 mL CMC oral)
EE.sub.2 100 .mu.g/kg, oral 215 .+-. 11 481 .+-. 24 raloxifene 1
mg/kg + 197 .+-. 7 263 .+-. 17 EE.sub.2 100 .mu.g/kg, oral
raloxifene 1 mg/kg 198 .+-. 11 202 .+-. 5
EXAMPLE 3
The ability of raloxifene to inhibit bone loss was compared to that
of tamoxifen (SIGMA, St. Louis, Mo.). Tamoxifen, a well known
antiestrogen currently used in the treatment of certain cancers,
has been shown to inhibit bone loss (see for example, Love, R., et
al. 1992 "Effects of tamoxifen on bone mineral density in
postmenopausal women with breast cancer", N. Eng J Med 326:852;
Turner, R., et al. 1988 "Tamoxifen inhibits osteoclast-mediated
resorption of trabecular bone in ovarian hormone-deficient rats",
Endo 122:1146). A relatively narrow range of doses of raloxifene
and tamoxifen was administered orally to ovariectomized rats as in
the previous example. Although both of these agents displayed the
ability to prevent reduction of femur density while evoking only
modest uterotrophic activity, as identified by gains in uterine
weight (Table 5), a comparison of several histological parameters
demonstrated a marked difference between the rats treated with
these agents (Table 6).
Increases is epithelial height are a sign of estrogenicity of
therapeutic agents and may be associated with increased incidence
of uterine cancer. When raloxifene was administered as described in
Example 1, only at one dose was there any statistically measurable
increase in epithelial height over the ovariectomized controls.
This was in contrast to the results seen with tamoxifen and
estrogen. At all doses given, tamoxifen increased epithelial height
equal to that of an intact rat, about a six-fold increase over the
response seen with raloxifene. Estradiol treatment increased
epithelial height to a thickness greater than intact rats.
Estrogenicity was also assessed by evaluating the adverse response
of eosinophil infiltration into the stromal layer of the uterus
(Table 6). Raloxifene did not cause any increase in the number of
eosinophils observed in the stromal layer of ovariectomized rats
while tamoxifen caused a significant increase in the response.
Estradiol, as expected, caused a large increase in eosinophil
infiltration.
Little or no difference was detectable between raloxifene and
tamoxifen effects on thickness of the stroma and myometrium. Both
agents caused an increase in these measurements that was much less
than the effect of estrogen.
A total score of estrogenicity, which was a compilation of all four
parameters, showed that raloxifene was significantly less
estrogenic than tamoxifen.
TABLE-US-00013 TABLE 5 Bone Density Uterine Weight (mg/cm/cm) (mg)
Ovariectomy control 171 .+-. 5 126 .+-. 17 (0.5 mL CMC oral) Intact
control 208 .+-. 4 490 .+-. 6 (0.5 mL CMC oral) EE.sub.2 100
.mu.g/kg, oral 212 .+-. 10 301 .+-. 37 raloxifene 1 mg/kg, oral 207
.+-. 13 198 .+-. 9 tamoxifen 1 mg/kg, oral 204 .+-. 7 216 .+-.
18
TABLE-US-00014 TABLE 6 Epithelial Stromal Myometrial Stromal Height
Eosinophils Thickness Expansion Ovariectomy control 1.24 1.00 4.42
10.83 (0.5 mL CMC oral) Intact control 2.71 4.17 8.67 20.67 (0.5 mL
CMC oral) EE.sub.2 100 .mu.g/kg, oral 3.42 5.17 8.92 21.17
raloxifene 1 mg/kg 1.67 1.17 5.42 14.00 tamoxifen 1 mg/kg 2.58 2.83
5.50 14.17
EXAMPLE 4
Other compounds of formula I were administered orally in the rat
assay described in Example 1. Table 7 reports the effect of a 1
mg/kg dose of each compound in terms of a percent inhibition of
bone loss and percent uterine weight gain.
TABLE-US-00015 TABLE 7 Compound % Inhibition % Uterine Number of
Bone Loss.sup.a Weight Gain.sup.b 2 86 26 6 24 19 8 66 24 10 52 24
11 26 28 12 60 15 14 121 32 16 108 25 18 21 17 27 25 1 34 26 -6
.sup.aPercent Inhibition of bone loss = (bone density of treated
ovex animals - bone density of untreated ovex animals) + (bone
density of estrogen treated ovex animals - bone density of
untreated ovex animals) .times. 100. .sup.bPercent uterine weight
gain = (uterine weight of treated ovex animals - uterine weight of
ovex animals) + (uterine weight of estrogen treated ovex animals -
uterine weight of ovex animals) .times. 100.
EXAMPLE 5
Fracture rate as a consequence of osteoporosis is inversely
correlated with bone mineral density. However, changes in bone
density occur slowly, and are measured meaningfully only over many
months or years. It is possible, however, to demonstrate that the
formula I compounds, such as raloxifene, have positive effects on
bone mineral density and bone loss by measuring various quickly
responding biochemical parameters that reflect changes in skeletal
metabolism. To this end, in a current test study of raloxifene at
least one hundred-sixty patients are enrolled and randomized to
four treatment groups; estrogen, two different doses of raloxifene,
and placebo. Patients are treated daily for eight weeks.
Blood and urine are collected before, during, and at the conclusion
of treatment. In addition, an assessment of the uterine epithelium
is made at the beginning and at the conclusion of the study.
Estrogen administration and placebo serve as the positive and
negative controls, respectively.
The patients are healthy post-menopausal (surgical or natural)
women, age 45-60 who would normally be considered candidates for
estrogen replacement in treatment for osteoporosis. This includes
women with an intact uterus, who have had a last menstrual period
more than six months, but less than six years in the past.
Patients who have received any of the following medications
systematically at the beginning of the study are excluded from the
study: vitamin D, corticosteroids, hypolipidemics, thiazides,
antigout agents, salicylates, phenothiazines, sulfonates,
tetracyclines, neomycin, and antihelmintics. Patients who have
received any estrogen, progestin, or androgen treatment more
recently than three months prior to the beginning of the study;
patients who have ever received calcitonin, fluoride, or
bisphosphonate therapy; patients who have diabetes mellitus;
patients who have a cancer history .[.anytime.]. .Iadd.any time
.Iaddend.within the previous five years; patients with any
undiagnosed or abnormal genital bleeding; patients with active, or
a history of, thromboembolic disorders; patients who have impaired
liver or kidney function; patients who have abnormal thyroid
function; patients who are poor medical or psychiatric risks; or
patients who consume an excess of alcohol or abuse drugs.
Patients in the estrogen treatment group receive 0.625 mg/day and
the two raloxifene groups receive dosages of 200 and 600 mg/day,
all groups receiving oral capsule formulations. Calcium carbonate,
648 mg tablets, is used as calcium supplement with all patients
taking 2 tablets each morning during the course of the study.
The study is a double-blind design. The investigators and the
patients do not know the treatment group to which the patient is
assigned.
A baseline examination of each patient includes quantitative
measurement of urinary calcium, creatinine, hydroxyproline, and
pyridinoline crosslinks. Blood samples are measured for serum
levels of osteocalcin, bone-specific alkaline phosphatase,
raloxifene, and raloxifene metabolites. Baseline measurements also
include examination of the uterus including uterine biopsy.
During subsequent visits to the investigating physician,
measurements of the above parameters in response to treatment are
repeated. The biochemical markers listed above that are associated
with bone resorption have all been shown to be inhibited by the
administration of estrogen as compared to an untreated individua.
Raloxifene is also expected to inhibit the markers in estrogen
deficient individuals as an indication that raloxifene is effective
in inhibiting bone loss from the time that treatment is begun.
Subsequent longer term studies can incorporate the direct
measurement of bone density by the use of a photon absorptiometry
and the measurement of fracture rates associated with therapy.
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