Note: Descriptions are shown in the official language in which they were submitted.
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CALCILYTIC COMPOUNDS
FIELD OF INVENTION
The present invention relates to the treatment of a variety of diseases
associated with abnormal bone or mineral homeostasis, including but not
limited to
hypoparathyroidism, osteosarcoma, periodontal disease, fracture healing,
osteoarthritis, rheumatoid arthritis, Paget's disease and osteoporosis. The
present
methods involve the co-administration of an orally active antagonist of the
calcium
receptor with an anti-resorptive agent.
In mammals, extracellular Ca2+ is under tight homeostatic control and
regulates various processes such as blood clotting, nerve and muscle
excitability,
and cellular function. Extracellular Ca2+ inhibits the secretion of
parathyroid
hormone ("PTH") from parathyroid cells, inhibits bone resorption by
osteoclasts,
and stimulates secretion of calcitonin from thyroid C-cells. Calcium receptor
proteins enable certain specialized cells to respond quickly to changes in
extracellular Ca2+ concentration.
PTH is the principal endocrine factor regulating Ca2+ homeostasis in the
blood and extracellular fluids. PTH, by acting on bone and kidney cells,
increases
the level of Ca2+ in the blood. This increase in extracellular Ca2+ acts as a
negative
feedback signal, depressing PTH secretion. The reciprocal relationship between
extracellular Ca2+ and PTH secretion forms an important mechanism maintaining
bodily Ca2+ homeostasis.
Extracellular Ca2+ acts directly on parathyroid cells to regulate PTH
secretion. The existence of a parathyroid cell surface protein which detects
changes
in extracellular Ca2+ has been confirmed. See Brown et al., Nature 366:574,
1993.
In parathyroid cells, this protein, the calcium receptor, acts as a receptor
for
extracellular Ca2+, detects changes in the ion concentration of extracellular
Ca2+,
and initiates a functional cellular response, PTH secretion.
Extracellular Ca2+ influences various cell functions, reviewed in Nemeth et
al., Cell Calcium 11:319, 1990. For example, extracellular Ca2+ plays a role
in
parafollicular (C-cells) and parathyroid cells. See Nemeth, Cell Calcium
11:323,
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1990. The role of extracellular Ca2+ on osteoclasts has also been studied. See
Zaidi,
Bioscience Reports 10:493, 1990.
Various compounds are known to mimic the effects of extra-cellular Ca2+ on
calcium receptors. Calcilytics are compounds able to antagonize calcium
receptor
activity, thereby causing a decrease in one or more calcium receptor
activities
evoked by extracellular Ca2+. Calcilytics are useful as lead molecules in the
discovery, development, design, modification and/or construction of calcium
receptor modulators which are active at Ca2+ receptors. Such calcilytics are
useful
in the treatment of various disease states characterized by abnormal levels of
one or
more components, e.g., polypeptides such as hormones, enzymes or growth
factors,
the expression and/or secretion of which is regulated or affected by activity
at one or
more Ca2+ receptors. Target diseases or disorders for calcilytic compounds
include
diseases involving abnormal bone and mineral metabolisim.
Abnormal calcium homeostasis is characterized by one or more of the
following activities: an abnormal increase or decrease in serum calcium; an
abnormal increase or decrease in urinary excretion of calcium; an abnormal
increase
or decrease in bone calcium levels (for example, as assessed by bone mineral
density
measurements); an abnormal absorption of dietary calcium; an abnormal increase
or
decrease in the production and/or release of messengers which affect serum
calcium
levels such as PTH and calcitonin; and an abnormal change in the response
elicited
by messengers which affect serum calcium levels.
Thus, calcium receptor antagonists offer a unique approach towards the
pharmacotherapy of diseases associated with abnormal bone or mineral
homeostasis,
such as hypoparathyroidism, osteosarcoma, periodontal disease, fracture
healing,
osteoarthritis, rheumatoid arthritis, Paget's disease and osteoporosis.
It is well known that chronic elevation of PTH, such as that seen in
hyperparathyroidism, leads to osteoclast-mediated bone loss and abnormal bone
histology. Dobnig and Turner, Endocrinol., Vol. 138, pp. 4607-4612 (1997),
showed that subcutaneous infusion of high doses of PTH (40 and 80 ug/kg/day)
over
periods of 2 hours or more led to rapid loss in body weight, hypercalcemia and
histological abnormalities in the skeleton consistent with changes seen in
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hyperparathyroidism. The literature indicates that, while intermittent
administration
of PTH is desirable in effecting bone formation, if the PTH elevation is too
prolonged, bone resorption is elevated. This limitation in the duration of PTH
elevation limits the choice of compounds that could be used to antagonize the
calcium receptor.
Therefore, there exists a need in the industry for a therapy that could
utilize
calcium receptor antagonists which might elicit transient PTH elevation
without the
concomitant resorption problems evidenced in the literature.
There is a further need for a therapy that causes relatively low degrees of
PTH elevation, while having the same beneficial effects as the treatments
currently
available.
SUMMARY OF THE INVENTION
The present invention provides novel methods of treatment of a variety of
diseases associated with abnormal bone or mineral homeostasis, including but
not
limited to hypoparathyroidism, osteosarcoma, periodontal disease, fracture
healing,
osteoarthritis, rheumatoid arthritis, Paget's disease and osteoporosis.
The present methods involve the co-administration of a calcilytic agent with
an anti-resorptive agent to a patient in need of treatment. The present
calcilytic
agents include agents which may cause prolonged PTH elevation. Preferably, the
present agents cause a transient elevation of PTH.
DETAILED DESCRIPTION OF DRAWING
Figure 1 represents proximal tibial BMD in osteopenic rats following treatment
with
calcilytic or PTH according to Study 1.
Seven month old rats were ovx and allowed to develop osteopenia for two
months.
Sham operated rats were treated with vehicle (0), ovx rats were treated with
vehicle
(o), NPS 2143 100umol/kg p.o. (a), or rat PTH 1-34 Sug/kg s.c. (0). BMD was
measured at the time points indicated. Statistical significance is indicated:
P<0.05; **P<0.01
Figure 2 represents plasma PTH levels in osteopenic rats treated with
calcilytic or rat
PTH according to Study 1.
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Timed plasma samples were collected relative to administration of the agent as
indicated, following treatment with calcilytic adminstered (filled circle) or
PTH
(open circle).
Figure 3 represents circulating levels of calcilytic following administration
of
calcilytic according to Study 1.
Timed plasma samples were collected relative to administration of the compound
as
indicated.
Figure 4 represents dynamic histomorphometry on proximal tibiae from
osteopenic
rats following treatment with calcilytic or PTH according to Study 1.
Figure 4a) represents % labeled perimeter (%L.Pm.)
Figure 4b) represents % eroded surface (% Er.P)
Figure 4c) represents % osteoid perimeter (%Os.Pm)
Figure 4d) represents bone formation rate: bone area referent (BFR/B.Ar)
%/year
Statistical significance is indicated: * P<0.05; **P<0.01
Figure 5 represents sections of tibiae stained with Von Kossa from osteopenic
ovx
rats treated with estrogen +/- calcilytic according to Study 2.
Representative sections are shown from animals treated for the next 5 weeks
with:
Figure Sa) represents vehicle
Figure Sb) represents 17(3 estradiol (s.c. pellet 0.01 mg/90 days)
Figure Sc) represents NPS 2143 ( 100 umol/kg daily p.o.)
Figure 6 represents histomorphometry on proximal tibiae from osteopenic rats
following treatment with calcilytic plus/minus 173 estradiol according to
Study 2.
Figure 6a) represents % trabecular bone area (%Tb.Ar)
Figure 6b) represents bone formation rate: tissue area referent (BFR/T.Ar)
%/year
Statistical significance is indicated: * P<0.05; **P<0.01
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DETAILED DESCRIPTION OF THE INVENTION
The calcilytic compounds of the present invention include all calcilytic
compounds. By "calcilytic compound", it is meant that the compound is able to
inhibit calcium receptor activity, thereby causing a decrease in one or more
calcium
receptor activities evoked by extracellular Ca2+. Such compounds include, but
are
not limited to a compound selected from the group consisting of:
N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine hydrochloride;
N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(4-
methoxyphenyl)ethyl amine hydrochloride;
N-[(2R-Hydroxy-3-[(2,3-dichloro)phenoxy-propyl]-1,1-dimethyl-2-(4-
methoxyphenyl)ethyl amine hydrochloride;
N-[(R)-2-Hydroxy-3-[2-cyano-4-[N-methyl-N-[3-carboxyphenyl)sulfonyl]amino]-
phenoxy]propyl]-1,1-dimethyl-2-(6-( 1,2,3,4-tetrahydronaphthyl)ethylamine;
N-[(R)-2-Hydroxy-3-[2-cyano-4-[N-methyl-N-[3-carboxyphenyl)sulfonyl]amino]-
phenoxy]propyl]-1,1-dimethyl-2-(Benzothien-3-yl)-ethylamine;
N-[(R)-2-Hydroxy-3-[2-cyano-4-[N-methyl-N-[3-carboxyphenyl)sulfonyl]amino]-
phenoxy]propyl]-1,1-dimethyl-2-(Benzothien-2-yl)-ethylamine;
N-[(R)-2-Hydroxy-3-[2-cyano-4-[N-methyl-N-[3-carboxyphenyl)sulfonyl]amino]-
phenoxy]propyl]-l,l-dimethyl-2-(decahydronapthalen-2-yl)ethylamine;
N-[(R)-2-Hydroxy-3-[2-cyano-4-[N-methyl-N-[3-carboxyphenyl)sulfonyl]amino]-
phenoxy]propyl]-1,1-dimethyl-4-phenylbutylamine;
N-[(R)-2-Hydroxy-3-[2-cyano-4-[N-methyl-N-[3-carboxyphenyl)sulfonyl]amino]-
phenoxy]propyl]-1,1-dimethyl-4-(2-methoxyphenyl)butylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[N-methyl-N-[4-ethylcarboxyphenyl]sulfonyl]-
amino]phenoxy]propyl]-1,1-dimethyl-2-(2-napthyl)ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[N-methyl-N-[3-
methylcarboxymethoxyphenyl]sulfonyl]-
amino]phenoxy]propyl]-1,1-dimethyl-2-(2-napthyl)ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methylsulfonyl]-N-[[[1-[2-[6-methyl]amino]-
pyridyl]ethyl]amino]phenoxy]propyl]-1,1-dimethyl-2-[2-napthyl]ethylamine;
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N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methylsulfonyl]-N-[[[ 1-[2-[6-methyl]amino]-
pyridyl]ethyl]amino]phenoxy]propyl]-1,1-dimethyl-2-( 1,2,3,4-tetrahydronaphth-
6-
yl)ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methylsulfonyl]-N-[[[1-[2-[6-methyl]amino]-
pyridyl]ethyl]amino]phenoxy]propyl]-1,1-dimethyl-2-(benzothien-3-yl)-
ethylamine;
N-[2R-Hydroxy-3-[ [2-cyano-4-[ [N-methylsulfonyl]-N-[ [ [ 1-[2-[6-
methyl]amino]-
pyridyl]ethyl]amino]phenoxy]propyl]-1,1-dimethyl-2-(benzothien-2-yl)-
ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methylsulfonyl]-N-[[[ 1-[2-[6-methyl]amino]-
pyridyl]ethyl] amino]phenoxy]propyl]-1,1-dimethyl-2-(decahydronapthalen-2-yl)-
ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methylsulfonyl]-N-[[[ 1-[2-[6-methyl]amino]-
pyridyl]ethyl]amino]phenoxy]propyl]-1,1-dimethyl-4-(2-
methoxyphenyl)butylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methylsulfonyl]-N-[[[ 1-[2-[6-methyl]amino]-
pyridyl]ethyl]amino]phenoxy]propyl]-l,1-dimethyl-4-phenylbutylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[N-benzyl-N-[4-methylphenyl]sulfonyl]amino]
phenoxy]propyl]-1,1-dimethyl-2-[4-methoxyphenyl]ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[N-[4-benzyl]sulfonyl]amino]
phenoxy]propyl]-1,1-dimethyl-2-[2-napthyl]ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-5-[[4-carboxy]phenyl]phenoxy]propyl]-
1,1-dimethyl-2-[napthyl]ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-[[N-methyl-N-[3-carboxyl]phenyl]sulfonyl]amino]-
phenoxy]propyl]-1,1-dimethyl-2-[2-napthyl]ethylamine; N-[2R-Hydroxy-3-[[2-
cyano-4-[[N-methyl-N-[3-
methylcarboxyl]phenyl]sulfonyl]amino]phenoxy]propyl]-1,1-dimethyl-2-[2-
napthyl]ethylamine;
N-[2R-Hydroxy-3-[ [2-cyano-4-(2-phenyl-2-R,S-carboxyl)phenoxy]-propyl]-1,1-
dimethyl-2-(2-naphthyl)ethylamine;
N-[2R-Hydroxy-3-[[2-cyano-4-(3-carboxypropyl)phenoxy]-propyl]-1,1-dimethyl-2-
naphthylethylamine;
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(N-[2R-Hydroxy-3-[[2-cyano-5-(3-carboxypropyl)phenoxy]-propyl]-1,1-dimethyl-2-
naphthylethylamine; and
(N-[2R-Hydroxy-3-[2-[2-[6-aminomethyl]pyridyl]ethyloxy]-l, l-dimethyl-2-
naphthylethylamine.
Bone is composed of a protein matrix in which spindle- or plate-shaped
crystals of hydroxyapatite are incorporated. Type I Collagen represents the
major
structural protein of bone comprising approximately 90% of the structural
protein.
The remaining 10% of matrix is composed of a number of non-collagenous
proteins,
including osteocalcin, proteoglycans, osteopontin, osteonectin,
thrombospondin,
fibronectin, and bone sialoprotein. Skeletal bone undergoes remodeling at
discrete
foci throughout life. These foci, or remodeling units, undergo a cycle
consisting of a
bone resorption phase followed by a phase of bone replacement.
Bone resorption is carried out by osteoclasts, which are multinuclear cells of
hematopoietic lineage. The osteoclasts adhere to the bone surface and form a
tight
sealing zone, followed by extensive membrane ruffling on their apical (i.e.,
resorting) surface. This creates an enclosed extracellular compartment on the
bone
surface that is acidified by proton pumps in the ruffled membrane, and into
which
the osteoclast secretes proteolytic enzymes. The low pH of the compartment
dissolves hydroxyapatite crystals at the bone surface, while the proteolytic
enzymes
digest the protein matrix. In this way, a resorption lacuna, or pit, is
formed. At the
end of this phase of the cycle, osteoblasts lay down a new protein matrix that
is
subsequently mineralized. In several disease states, such as osteoporosis and
Paget's
disease, the normal balance between bone resorption and formation is
disrupted, and
there is a net loss of bone at each cycle. Ultimately, this leads to weakening
of the
bone and may result in increased fracture risk with minimal trauma.
As used herein "anti-resorptive" means an agent capable of preventing,
delaying or retarding bone resorption. Anti resorptive agents useful in the
present
invention include, but are not limited to, estrogen, 1, 25 (0H)2 vitamin D3,
calcitonin, bisphosphonate and cathepsin K inhibitors.
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The present compounds can also be formulated as pharmaceutically
acceptable salts and complexes thereof. Pharmaceutically acceptable salts are
non-
toxic salts in the amounts and concentrations at which they are administered.
Pharmaceutically acceptable salts include acid addition salts such as those
containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate,
acetate,
citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-
toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable
salts can be obtained from acids such as hydrochloric acid, malefic acid,
sulfuric acid,
phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid,
tartaric acid,
malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,
p-
toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic addition salts such as
those containing benzathine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium,
potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional
groups, such as carboxylic acid or phenol are present.
In order to use a compound of the present invention or a pharmaceutically
acceptable salt thereof for the treatment of humans and other mammals, it is
normally formulated in accordance with standard pharmaceutical practice as a
pharmaceutical composition.
The calcilytic compounds can be administered by different routes including
intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical
(transdermal),
or transmucosal administration. For systemic administration, oral
administration is
preferred. For oral administration, for example, the compounds can be
formulated
into conventional oral dosage forms such as capsules, tablets, and liquid
preparations
such as syrups, elixirs, and concentrated drops.
Alternatively, injection (parenteral administration) may be used, e.g.,
intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection,
the
compounds of the invention are formulated in liquid solutions, preferably, in
physiologically compatible buffers or solutions, such as saline solution,
Hank's
solution, or Ringer's solution. In addition, the compounds may be formulated
in
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solid form and redissolved or suspended immediately prior to use. Lyophilized
forms can also be produced.
Systemic administration can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants appropriate to the
barrier
to be permeated are used in the formulation. Such penetrants are generally
known in
the art, and include, for example, for transmucosal administration, bile salts
and
fusidic acid derivatives. In addition, detergents may be used to facilitate
permeation.
Transmucosal administration, for example, may be through nasal sprays, rectal
suppositories, or vaginal suppositories.
For topical administration, the compounds of the invention can be formulated
into ointments, salves, gels, or creams, as is generally known in the art.
The amounts of various calcilytic compounds to be administered can be
determined by standard procedures taking into account factors such as the
compound
IC50, EC50, the biological half-life of the compound, the age, size and weight
of the
patient, and the disease or disorder associated with the patient. The
importance of
these and other factors to be considered are known to those of ordinary skill
in the
art.
Amounts administered also depend on the routes of administration and the
degree of oral bioavailability. For example, for compounds with low oral
bioavailability, relatively higher doses will have to be administered.
Preferably the composition is in unit dosage form. For oral application, for
example, a tablet, or capsule may be administered, for nasal application, a
metered
aerosol dose may be administered, for transdermal application, a topical
formulation
or patch may be administered and for transmucosal delivery, a buccal patch may
be
administered. In each case, dosing is such that the patient may administer a
single
dose.
Each dosage unit for oral administration contains suitably from 0.01 to 500
mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof, calculated as the free base. The
daily
dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal
routes
contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(I). A
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topical formulation contains suitably 0.01 to S.o% of a compound of Formula
(I).
The active ingredient may be administered from 1 to 6 times per day,
preferably
once, sufficient to exhibit the desired activity, as is readily apparent to
one skilled in
the art.
As used herein, "treatment" of a disease includes, but is not limited to
prevention, retardation and prophylaxis of the disease.
Diseases and disorders which might be treated or prevented, based upon the
affected cells, include bone and mineral-related diseases or disorders;
hypoparathyroidism; those of the central nervous system such as seizures,
stroke,
head trauma, spinal cord injury, hypoxia-induced nerve cell damage, such as
occurs
in cardiac arrest or neonatal distress, epilepsy, neurodegenerative diseases
such as
Alzheimer's disease, Huntington's disease and Parkinson's disease, dementia,
muscle
tension, depression, anxiety, panic disorder, obsessive-compulsive disorder,
post-
traumatic stress disorder, schizophrenia, neuroleptic malignant syndrome, and
Tourette's syndrome; diseases involving excess water reabsorption by the
kidney,
such as syndrome of inappropriate ADH secretion (SIADH), cirrhosis, congestive
heart failure, and nephrosis; hypertension; preventing and/or decreasing renal
toxicity from cationic antibiotics (e.g., aminoglycoside antibiotics); gut
motility
disorders such as diarrhea and spastic colon; GI ulcer diseases; GI diseases
with
excessive calcium absorption such as sarcoidosis; autoimmune diseases and
organ
transplant rejection; squamous cell carcinoma; and pancreatitis.
In a preferred embodiment of the present invention, the present compounds
are used to increase serum parathyroid ("PTH") levels in a non-pulsatile
manner.
Increasing serum PTH levels may be helpful in treating diseases such as
hypoparathyroidism, osteosarcoma" periodontal disease, fracture,
osteoarthritis,
rheumatoid arthritis, Paget's disease and osteoporosis.
The normal range for intact PTH in humans is about 10 to about 65 pg/ml.
Increasing serum PTH may also be useful to prophylactically retard or prevent
the
onset of a disease. Prophylactic treatment can be performed, for example, on a
person with a low serum PTH, or a person without low serum PTH, but where
increasing PTH has a beneficial compensating effect. Preferably, the patient
has an
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abnormally low serum PTH. As used herein, "abnormally low serum PTH" means a
serum PTH level lower than that occurring in the general population, and is
preferably an amount associated with a disease or onset of a disease.
Increasing serum PTH levels can be used to treat various diseases including
bone and mineral related diseases.
Preferably, the duration of PTH level increase is 12 hours or longer, more
preferably 18 hours or longer and most preferably 24 hours or longer.
Preferably, the increase in PTH is 3 fold or lower than the normal range for
intact PTH in humans. More preferably, the increase in PTH level is 2-fold or
lower
than the normal range.
The present invention also provides compositions comprising the present
compounds and their pharmaceutically acceptable salts which are active when
given
orally can be formulated as syrups, tablets, capsules and lozenges. A syrup
formulation will generally consist of a suspension or solution of the compound
or
salt in a liquid carrier for example, ethanol, peanut oil. olive oil,
glycerine or water
with a flavoring or coloring agent. Where the composition is in the form of a
tablet,
any pharmaceutical carrier routinely used for preparing solid formulations may
be
used. Examples of such carriers include magnesium stearate, terra alba, talc,
gelatin,
acacia, stearic acid, starch, lactose and sucrose. Where the composition is in
the
form of a capsule, any routine encapsulation is suitable, for example using
the
aforementioned carriers in a hard gelatin capsule shell. Where the composition
is in
the form of a soft gelatin shell capsule any pharmaceutical carrier routinely
used for
preparing dispersions or suspensions may be considered, for example aqueous
gums,
celluloses, silicates or oils, and are incorporated in a soft gelatin capsule
shell.
Typical parenteral compositions consist of a solution or suspension of a
compound or salt in a sterile aqueous or non-aqueous carrier optionally
containing a
parenterally acceptable oil, for example polyethylene glycol,
polyvinylpyrrolidone,
lecithin, arachis oil or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension
or emulsion that may be administered as a dry powder or in the form of an
aerosol
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using a conventional propellant such as dichlorodifluoromethane or
trichlorofluoromethane.
A typical suppository formulation comprises a compound of the present
invention or a pharmaceutically acceptable salt thereof which is active when
administered in this way, with a binding and/or lubricating agent, for example
polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes
or
fats or their synthetic analogs.
Typical dermal and transdermal formulations comprise a conventional
aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste
or
are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet,
capsule or metered aerosol dose, so that the patient may administer a single
dose.
No unacceptable toxological effects are expected when compounds of the
present invention are administered in accordance with the present invention.
Biological Assays:
The following assays were performed.
Ovariectomized Rat Studies
Study 1
Seven month old virgin Sprague Dawley female rats were subjected to
bilateral ovariectomy or sham surgery and the animals then held for a period
of three
months to allow the development of osteopenia. At that time a single sham
group
(n=10) and three groups of ovariectomized (ovx) animals (n = 10 - 14) were
assigned. The ovx groups were selected such that there was no significant
difference
in bone mineral density ('°BMD") of the lumbar spine, proximal tibia or
distal femur
between groups. Groups consisted of sham and ovx controls treated with dose
vehicle (20% aqueous encapsin) and ovx groups treated with either N-[(2R-
Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine hydrochloride ("calcilytic administered") ( 100 umol/kg
body
weight daily p.o.) or rat PTH 1-34 (5 ug/kg body weight daily s.c.).
During the study blood samples were drawn for determination of circulating
PTH and osteocalcin. BMD was determined by DXA (QDR-4500 Hologic,
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Waltham, Mass) prior to treatment and at weeks 4 and 8. At term tibiae were
removed for histological analysis. All animals received tetracycline 10 and 3
days
prior to the start of dosing and calcein ( 10 mg/kg) 10 and 3 days prior to
sacrifice.
Study 2
Animals were prepared and monitored as described above. Groups consisted
of sham and ovx controls which were treated with oral dose vehicle (20%
aqueous
encapsin), and 4 additional ovx groups that received either N-[(2R-Hydroxy-3-
[(3-
chloro-2-cyano)phenoxy-propyl]-l,1-dimethyl-2-(2-naphthyl)ethyl amine
hydrochloride(100 umol/kg/d, p.o.), 17(3 estradiol (c.c. pellet 0.01
mg/90days), or
N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine hydrochloride+ estradiol (each as above). Dosing
continued
for 5 weeks at which point the animals were sacrificed and the tibiae were
collected
for histological analysis.
Measurement of circulating compound and PTH levels
Timed plasma samples were collected relative to administration of
compound or PTH. PTH 1-34 was measured by RIA (Nichols Institute Diagnostics,
San Juan Capistrano, CA). The concentrations of compound in the plasma were
quantified by LC/MS/MS (limit of detection = 10 ng/ml).
Histomorphometric evaluation
Bone samples were dehydrated through increasing concentrations of ethanol,
defatted in acetone and embedded in methyl methacrylate (Polysciences, Inc.,
Warrington, PA). Longitudinal undecalcified sections (5 Vim) sections of the
proximal tibial were cut on a Leica microtome (SM2500S); the tissue blocks had
been prestained with Villanueva stain. Histomorphometric analysis was carried
out
using an Osteomeasure system (OsteoMetrics Incorpotated), without knowledge of
group allocation. Measurements within the tibial metaphysic were restricted to
a
mean tissue area of approximately 8 mmz beginning 1 mm below the growth plate.
Primary measurements included area of bone and marrow (mm2), bone area (mm2),
perimeter of bone (mm), single and double-labelled perimeter (sL.Pm, dL.Pm,
mm),
osteoid perimeter (O.Pm, mm) and eroded perimeter (Er.P, mm). Derived indices
included trabecular bone volume (%Tb.Ar), trabecular number (Tb.N, mrri'),
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trabecular thickness (Tb.Th, Vim), trabecular separation (Tb.Sp, Vim), bone
formation
rate, surface referent (BFR/Tb.Pm, ~tm3/~m2/year), BFR/Tb.Ar (bone area
referent,
%/year), BFR/B.Ar (tissue referent, %/year) and mineral apposition rate (MAR,
~tm/day), and percent labelled perimeter (%Lp). Statistical analysis was
assessed by a
two sided t-test.
Human osteoclast-mediated bone resorption assay
The isolation of disaggregated human osteoclasts from fresh osteoclastoma
tissue and the in vitro human osteoclast resorption assay were performed
according
to James, J. Bone Min. Res., Vol. 1 l, pp.1453-1460. Briefly, human
osteoclasts
were seeded onto bovine cortical bone particles with compound or vehicle for
24
hours at 37 °C. The culture media were then removed and the levels of
the carboxy-
terminal peptide of the al chain of human type I collagen were quantified as a
biochemical readout of resorption, using a competitive binding enzyme linked
immunosorbant assay (ELISA) ( 19) (Osteometer A/S, Rodovre, Denmark). The
results are expressed as percent inhibition of resorption compared to
supernatants
derived from osteoclasts cultured in vehicle without inhibitor. IC;" values
are
determined from the resultant dose response curves.
Fetal Rat Long Bone Resorption Assay
The assay was performed essentially as in Votta, Bone, Vol. 15, pp. 533-538,
( 1994). Timed-pregnant Sprague-Dawley rats (Taconic Farms, Germantown, NY)
were injected subcutaneously with 200 microcuries of 45CaC12 on day 18 of
gestation, housed overnight, then anesthetized with Innovar-Vet (Pittman-
Moore,
Mundelein, IL) and sacrificed by cervical dislocation. The fetuses were
removed
aseptically and the radii and ulnae were dissected free of surrounding soft
tissue and
cartilagenous ends. The bone rudiments (n = 4) were subsequently cultured for
18-24
hours in BGJb medium (Sigma, St. Louis, MO) containing 1 mg/ml BSA, then
transferred to fresh medium and cultured for an additional 48 hours in the
absence or
presence of PTH (human parathyroid hormone [1-34], Bachem, Torrence, CA) and
the desired inhibitor. 45Ca released into the medium and the residual 45Ca in
the
bones (following solubilization in 5% TCA for 1 hour at room temperature) were
quantitated by liquid scintillation spectrometry. Data are expressed as the
percent
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45Ca released from treated bones as compared with corresponding control bones.
Statistical differences were assessed by a one way analysis of variance
(ANOVA).
IC-50 values were based on data from two independent experiments.
Osteoblast cAMP production and alkaline nhosuhatase activity
cAMP accumulation was measured in both human TF274 osteoblastic cells
(derived by immortalization of human bone marrow stromal cells) see James, s-
upra,
and primary human osteoblasts derived from explants of trabecular bone as
described in Beresford, Biochim. Biophys. Acta, Vol. 801, pp. 58-65 (19884).
CAMP levels in cell samples were measured using a non-radioactive protocol
(Amersham kit). Alkaline phosphatase activity was determined using the
standard
colorimetric method as described previously in Gowen, Arth. Rheum., Vol. 31,
pp.
1500-1507 (1988). N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-
dimethyl-2-(2-naphthyl)ethyl amine hydrochloride was tested at 0.1, 1 and 10
uM.
PTH 40 ng/ml was used as a positive control.
Results obtained from the assays described in Study 1 indicate that small but
sustained elevation of PTH levels causes increased bone turnover with no net
bone
gain or loss.
Bone mineral density (BMD) was measured in vivo in the lumbar spine,
distal femur and proximal tibia immediately before treatment and following
eight
weeks of dosing. Animals which had been ovariectomized three months previously
had lost significant bone mass at all three skeletal sites: 15% at lumbar
spine and
proximal tibia, 24% at the distal femur. During the course of treatment bone
mass
was unaffected by treatment with N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-
propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine, but was returned to pre-ovx
levels
after eight weeks of treatment with Sug/kg daily PTH in the proximal tibia
(Figure
1 ). Measurement of plasma PTH levels at the end of this experiment showed
that
the animals which received N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-
1,1-dimethyl-2-(2-naphthyl)ethyl amine had elevated PTH levels (>100 pg/ml)
which remained high at four hours after administration of the drug (Figure 2).
It is
not known how long this elevation was sustained, although PTH levels were back
to
baseline after 24 hours (immediately prior to next dose). PTH levels in
animals
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given 5 ug/kg PTH were in the same range as those dosed with N-[(2R-Hydroxy-3-
((3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine, but were returned to baseline by 2-4 hours after dosing
(Figure 2). The differences in the duration of the PTH response can be
explained by
sustained exposure to N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-
dimethyl-2-(2-naphthyl)ethyl amine, which was found to be elevated for up to
eight
hours (Figure 3).
The difference in the PTH profile obtained under these two dosing conditions
has allowed us to determine directly the effect of time of exposure to PTH on
bone
turnover. Dynamic histomorphometry of the proximal tibia showed that bone
formation (%L.Pm., % Os.Pm) was elevated above the ovx control level by both
PTH and N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-
(2-naphthyl)ethyl amine, (Figure 4a, b). Mineral apposition rate was unchanged
by
any treatment. However bone resorption, as measured by % eroded perimeter, was
significantly higher in the N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-
propyl]-
l,1-dimethyl-2-(2-naphthyl)ethyl amine, group than in the PTH or ovx control
groups (figure 4c). This is exemplified further by the dramatic increase in
bone
turnover demonstrated by the BFR/B.Ar. in the N-[(2R-Hydroxy-3-[(3-chloro-2-
cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine-treated compared
with the other two groups (Figure 4d). Thus the modest but prolonged elevation
of
PTH achieved by administration of calcilytic administered resulted in a
dramatic
increase in both bone formation and resorption, with no net bone gain or loss.
PTH
administered exogenously also increased both resorption and formation, but
formation exceeded resorption, resulting in increased bone mass.
Results from Study 2 indicate that small but sustained elevation of PTH
levels in the presence of an anti-resorptive agent causes increased bone
turnover with
net bone gain.
A second study was performed in which N-[(2R-Hydroxy-3-[(3-chloro-2-
cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine hydrochloride was
administered daily for 4 weeks, in the presence or absence of estrogen to
seven
month old rats which had been ovx three months earlier. Figure 5 shows
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representative sections of tibiae stained with Von Kossa from animals with no
treatment following ovx (5a), treated with estrogen alone (5b) or treated with
estrogen plus N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-
2-(2-naphthyl)ethyl amine(Sc). It is clear that co-administration of N-[(2R-
Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl
amine and estrogen (5c) caused increased bone mass over and above estrogen
alone.
Static histomorphometry of the proximal tibia (Table 1) showed that %
trabecular
area (%Tb.Ar.) was 72% lower in ovx animals compared to sham (P<0.0001). This
bone loss was not significantly restored by estradiol. N-[(2R-Hydroxy-3-[(3-
chloro-
2-cyano)phenoxy-propyl]-l,1-dimethyl-2-(2-naphthyl)ethyl amine alone had no
effect on the ovx-induced osteopenia. However, N-[(2R-Hydroxy-3-[(3-chloro-2-
cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine plus estrogen
resulted in a two-fold increase in % Tb.Ar. over the ovx group (Figure 6a).
This
appears to be due to an increase in trabecular thickness induced by N-[(2R-
Hydroxy-
3-[(3-chloro-2-cyano)phenoxy-propyl]-l,1-dimethyl-2-(2-naphthyl)ethyl amine
plus
estrogen (Table 1 ). The bone formation rate/tissue area was significantly
elevated in
the N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine plus estrogen group (Figure 6b). Elevation of bone
formation
rate/tissue area shows that bone mass is increasing in the area measured and
reflects
new bone formation on bone surfaces that are not being remodeled (modeling), a
classic feature of PTH action. This appears to be a result of a decrease in
resorption
(presumably due to concurrent estrogen treatment) relative to the N-[(2R-
Hydroxy-
3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine -
treated animals in the face of maintained elevation of bone formation.
Direct effects of N-f (2R-Hydroxy-3-((3-chloro-2-cyano)phenoxy-propyll-1,1-
dimethyl-2-(2-naphthyl)ethyl amine on osteoblasts and osteoclasts in vitro
Since Ca2+ sensing receptors have been demonstrated on both osteoblasts
and osteoclasts we studied the direct effects of N-[(2R-Hydroxy-3-[(3-chloro-2-
cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine on both
osteoblasts
and osteoclasts in vitro were studied.
Osteoblast activity
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While PTH caused a two-fold increase in cAMP levels in both cell types
used, N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine - had no effect on basal or PTH-induced cAMP levels.
Treatment of TF274 cells with N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-
propyl]-l,1-dimethyl-2-(2-naphthyl)ethyl amine did not result in any change in
alkaline phosphatase activity, nor was PTH-induced alkaline phosphatase
affected
by N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine. N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-
dimethyl-2-(2-naphthyl)ethyl amine demonstrated some toxicity in vitro at the
10
uM concentration.
Osteoclast activity
N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine had no effect on human osteoclast mediated bone
resorption at
concentrations up to 3 uM, while an inhibitor of cathepsin K, 3,11-bis (2-
methylpropyl)-4,7,10-trioxo-2,5,6,8,9,12-hexaazatridecanedioate inhibited with
an
IC50 of 0.9 uM. This assay is limited by its sensitivity to DMSO, so
concentrations
of N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine above 3 uM could not be tested. In the fetal rat long
bone
assay N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-
naphthyl)ethyl amine inhibited resorption with an ICSp of 11.3 +/- 3 uM. The
mechanism of this inhibition is not understood and since the effect occurs at
concentrations approximately 300 fold higher than the IC50 for Ca2+ receptor-
mediated Ca2+ mobilization it may well be unrelated to any effect on the Ca2+
receptor. The possibility that it could be related to toxicity cannot be ruled
out.
The above experiments demonstrated that a small orally active compound
can be designed which induces endogenous PTH secretion sufficiently to
stimulate
bone turnover. The pharmacokinetic characteristics of this molecule are such
that a
prolonged elevation of PTH (>4 hours) is obtained. This has allowed us to
examine
the role of the duration of PTH elevation at low levels of circulating PTH.
When
PTH was elevated for greater than four hours bone turnover was further
elevated, but
remained in balance leading to no net loss or gain. The co-therapy experiment
was
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performed with estrogen and antagonist co-treatment resulting in an increase
in bone
formation as measured histomorphometrically.
It is well known that chronic elevation of PTH, such as that seen in
hyperparathyroidism, leads to bone loss and abnormal bone histology. Dobnig et
al.,
s. upra, showed that subcutaneous infusion of high doses of PTH (40 and 80
ug/kg/day) over periods of 2 hours or more led to rapid loss in body weight,
hypercalcemia and histological abnormalities in the skeleton consistent with
changes
seen in hyperparathyroidism. In our study the much smaller increases in PTH,
although sustained, did not lead to these adverse effects. However, the
anabolic
effect of PTH was still lost with sustained exposure. This suggests that a
mild
hyperparathyroid condition, whether natural or pharmacologically-induced,
could be
asymptomatic.
The present data also demonstrate that, with regard to the amount of PTH
secreted in response N-[(2R-Hydroxy-3-[(3-chloro-2-cyano)phenoxy-propyl]-1,1
dimethyl-2-(2-naphthyl)ethyl amine, a low duration and low fold increase in
PTH
levels had profound effects on bone turnover. Most published studies on
effects of
PTH in rats have used a dose of 80 ug/kg. This dose leads to a circulating
level of
approximately 5,000 - 14,000 pg/ml, compared to the 150-200 pg/ml in our
studies.
This demonstrates that very low doses of PTH effectively modulate bone
turnover.
This is also illustrated by the much lower doses used in the clinical studies
performed recently in which approximate doses of 0.4-0.8 ug/kg body weight led
to
increased bone mass (8,24). 0.4 ug/kg led to circulating levels of
approximately 90
pmol/1 of PTH 1-34 at 30 minutes after dosing (25). This is an approximately
three-
fold increase in circulating PTH levels. Thus it appears that the PTH stored
in the
parathyroid gland will be sufficient to cause an anabolic effect if released
in
response to a Ca2+ receptor antagonist.
The above data demonstrated for the first time that stimulation of
endogenous parathyroid hormone secretion using an antagonist of the
parathyroid
cell Ca2+ receptor results in increased bone formation and resorption. In the
presence of an anti-resorptive agent N-[(2R-Hydroxy-3-[(3-chloro-2-
cyano)phenoxy-propyl]-1,1-dimethyl-2-(2-naphthyl)ethyl amine, caused an
increase
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in bone mass. This provides the basis for the development of a novel class of
anabolic agent for the treatment of osteoporosis.
All publications, including but not limited to patents and patent applications
cited in this specification are herein incorporated by reference as if each
individual
publication were specifically and individually indicated to be incorporated by
reference as though fully set forth.
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