Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF INCREASING BONE VOLUME
TECHNICAL FIELD
The present invention relates to novel methods of increasing bone volume
comprising activating the osteoblastic protein kinase C/ intracellular calcium
pathways of
a subject. This invention further relates to a method of treating or
preventing bone
disorders wherein activation occurs by the administration of a FP agonist.
BACKGROUND OF THE INVENTION
In osteoporotics an imbalance in the bone remodeling process develops in which
bone is resorbed at a rate faster than it is being made. Although this
imbalance occurs to
some extent in most individuals, both male and female, as they age, it is much
more
severe and occurs at a younger age in osteoporotics, particularly those who
develop the
post menopausal form of the condition. Accelerated bone loss may also result
from drug
administration, such as corticosteroids; prolonged bedrest; disuse of a limb;
and
microgravity. A consequence of this loss of bone is the complete removal of
trabeculae
and a deterioration of bone architecture such that the remaining bone is
disproportionately decreased in strength.
It is thought that to completely return the bone to normal strength, new
trabeculae
should be formed to restore architecture and increase bone mass. It is further
thought
that when the restoration of normal architecture is associated not only with
an increase in
the strength, but also a return to normal stiffness and shock absorbing
capability, the
bone is less likely to fracture. Subjects suffering from other bone disorders
such as
osteoarthritis, Paget's disease, periodontal disease, and fractures may also
benefit from
treatments that restore bone mass and normal architecture to bone.
There have been many attempts to treat bone disorders with a variety of
pharmacologic compounds with the goal being to either slow further bone loss
or to
produce a net gain in bone mass. For example, there are antiresorptive agents,
such as
bisphosphonates, which only slow further bone loss. In addition, there are
known bone
anabolic agents such as PTH and PGEz. But, neither of these agents builds bone
that is
substantially similar, i.e. structurally or architecturally, to the type of
bone lost.
PTH and PGEz are known to stimulate both the cAMP and protein kinase C/
intracellular calcium pathways. In general, it is believed that stimulating
the cAMP
pathway is necessary, and may be sufficient, to build bone. This belief is
based, at least
in part, upon observations that PTH analogs that only increase cAMP increase
trabecular
thickness, while PTH analogs that only increase PKC/Ca++ do not increase bone
mass.
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This belief is further based on the observation that PGE, analogs that
selectively
stimulate the EPz receptor, which is coupled primarily to the cAMP pathway,
increase
bone volume. PCT Publication WO 98/27976.
In addition to building bone that is not substantially similar to the type of
bone
lost, these known anabolic agents such as PTH and PGEZ have several drawbacks
which
limit their desirability for systemic administration. For example, although
prostaglandins
are characterized by their activity at a particular prostaglandin receptor,
their activity is
not limited to any one prostaglandin receptor. Thus, systemic administration
of
prostaglandins is known to cause side effects such as inflammation, as well as
surface
irritation, smooth muscle contraction, bronchoconstriction, and
vasoconstriction.
Systemic administration of non-selective prostaglandin analogs can likewise
cause side
effects.
Thus, there is a continuing need to develop methods of replacing bone that
result
in bone that is substantially similar, structurally and architecturally, to
the type of bone
lost.
SUMMARY OF THE INVENTION
It has been unexpectedly found that agents which primarily activate the
osteoblastic protein kinase C/ intracellular calcium pathways significantly
improve bone
quality over other anabolic agents. Particularly preferred agents are agents
that are
selective for FP receptors. Particularly preferred agents selective for FP
receptors are
non-naturally-occurring FP agonist. Particularly preferred non-naturally-
occurring FP
agonists are selective for the FP receptor over other excitatory prostaglandin
receptors in
a ratio of at least about 1:10, more preferably at least about 1:20, and most
preferably at
least about 1:50. Still more preferred non-naturally-occurring FP agonists are
selective
for the FP receptor over all other prostanoid receptors in a ratio of at least
about 1:10,
more preferably at least about 1:20, and most preferably at least about 1:50.
It has been further found that agents which primarily activate the
osteoblastic
protein kinase C/ intracellular calcium pathways increase trabecular number
through
formation of new trabeculae, increase bone volume and mass while maintaining a
normalized bone turnover rate and increase formation at the endosteal surface
without
removing bone from the existing cortex.
Accordingly, the present invention is directed to methods of increasing bone
volume, increasing trabecular number, and treating bone disorders by primarily
activating the osteoblastic protein kinase C/ intracellular calcium pathways.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a representative drawing of the protein kinase C/ intracellular Ca~"
pathways.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to methods of increasing bone volume,
methods
of increasing trabecular number, and methods of treating bone disorders by
primarily activating the ostoeblastic protein kinase C/ intracellular calcium
pathways
("PKC/Ca++ pathways").
Definitions and Usage of Terms
As used herein, "activates a subject's ostoeblastic protein kinase C
/intracellular
calcium pathways" refers to the activation of the Gq family of proteins
associated with
seven transmembrane receptor proteins which initiates the intracellular
messenger
cascade containing both protein kinase C (PKC) and Ca+''.
As used herein, "anabolic agent" means an agent which increases bone.
As used herein, "bone disorder" means the need for bone repair or replacement.
Conditions in which the need for bone repair or replacement may arise include:
osteoporosis (including post menopausal osteoporosis, male and female senile
osteoporosis and corticosteroid induced osteoporosis), osteoarthritis, Paget's
disease,
osteomalacia, multiple myeloma and other forms of cancer, prolonged bed rest,
chronic
disuse of a limb, anorexia, microgravity, exogenous and endogenous gonadal
insufficiency, bone fracture, non-union, defect, prosthesis implantation and
the like.
As used herein, "bone turnover rate" means the amount of bone resorption and
formation per unit time measured or estimated using incorporation of
fluorescent labels
into bone, fluorescent and bright field microscopy, and histomorphometric
techniques or
by measurement of bone metabolism markers. For example, a subject may resorb
and
replace (turn over) approximately 3% of its skeleton over a 3 month period. A
further
description of histomorphometric techniques can be found in Bone
Histomorphometry,
1994, by Eriksen et. al., Raven Press.
As used herein, "bone volume" refers to the percentage of the bone occupied
by a mineralized matrix. Measurement or estimation of the mineralized matrix
volume
can be accomplished using histomorphometry, computed tomography, or magnetic
resonance imaging. Two dimensional measurements may be used to estimate the
three
dimensional volume. A further description of histomorphometric techniques can
be
found in Bone Histomorphometry, 1994, by Eriksen et. al., Raven Press.
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As used herein, "excitatory prostaglandin receptor" means prostanoid receptors
which cause contraction of smooth muscle or release of internal calcium
stores. Such
receptors include but are not limited to FP, EP,, EP3, TP, and TPz.
As used herein, "FP" is an abbreviation for F prostanoid.
As used herein, "FP agonist" means a compound with affinity for the FP
receptor
that results in measurable biological activity (including but not limited to
an elevation in
intracellular calcium or the contraction of smooth muscle) in cells, tissues,
or organisms
which contain the FP receptor. Whole cell, tissue, and organism assays which
demonstrate FP activity of compounds are well known in the art. One
particularly useful
assay is the R-SAT'"'' Assay described by Brann, et al. in J. Biomole. Screen,
Vol. 1,
Number 1, 1996.
As used herein, "FP receptor" means known human FP receptors, their splice
variants, and undescribed receptors that preferentially bind PGFza. A human FP
receptor
is disclosed in PCT Publication WO 95/00551.
As used herein, "measurable" means the biologic effect is both reproducible
and
significantly different from the baseline variability of the assay.
As used herein, "non-naturally-occurring" means an agent that is not
biologically
derived in mammals.
As used herein, "osteoblastic" refers to cells of the osteoblast lineage,
which
includes precursor or progenitor cells, pre-osteoblasts, osteoblasts, bone
lining cells and
osteocytes.
As used herein, "primarily" means the agent preferentially activates the
osteoblastic PKC/Ca++ pathways over the cAMP pathway. Preferential activation
of the
PKC/Ca++ pathways over the cAMP pathway can be measured using a variety of
assays.
For example, intracellular Ca++ concentration can be measured by the use of
the calcium
indicator Fura-2 assay described in "The molecular Biology of the Cell",
edited by
Alberts et al., Garland Publishing, 1994, p. 183, and intracellular cAMP can
be measured
by the assay described in "The principles of bone biology" edited by J
Bilezikian et aL,
Academic Press, 1996, p 1205. To provide a direct comparison of these two
pathways, a
ratio of activation of 1 is defined as 100% of the maximum activation of the
PKC/Ca++
pathways of PGEz divided by 100% of the activation of the CAMP pathway by
PGE2.
The concentrations of PGEZ required to reach the maximum activation may differ
by up
to 2 orders of magnitude. Preferential activation would then represent an
increase in this
ratio above about 1, preferably above about 2, more preferably above about
3.5, and most
preferably above about 5, where the pathway activator could either increase
the
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PKC/Ca~ pathway activation (for example to 125% of PGEZ) or decrease the cAMP
pathway activation (for example to 75% of PGE,).
As used herein, "prostaglandin analog" refers to a non-naturally-occurring
compound which is structurally similar to a prostaglandin.
As used herein, "prostaglandin receptor" or "prostanoid receptor" means a
naturally-occurring protein that binds prostaglandins, which when bound alters
the
function of a cell. Prostaglandin receptors may be characterized as either
excitatory or
relaxant. Such receptors include but are not limited to FP, EP,, EPZ, EP3,
EP4, DP, IP,
TP, and TP,. These receptors are further discussed by Coleman et al., in
Pharmacological Reviews, 1994, Volume 6, No. 2, pages 205 - 229.
As used herein, "selective" means having an activation preference for a
specific
receptor over other receptors which can be quantified based upon whole cell,
tissue, or
organism assays which demonstrate receptor activity, such as the R-SAT's''
Assay
disclosed above. A compound's selectivity is determined from a comparison of
its ECso
(or EDso if using an organism assay) at the relevant receptors. For example, a
compound
having an ECso of 8nM at the FP receptor and an ECso of 80 nM at the EP,
receptor has a
selectivity ratio for the FP receptor over the EP, receptor of 1:10.
As used herein, "subject" means a living vertebrate animal such as a mammal
(especially human) in need of treatment.
As used herein, "trabecular number" refers to the number of individual
trabeculae
of bone per unit volume of cancellous bone measured or estimated from a two
dimensional representation or a three dimensional specimen using
histomorphometry,
computed tomography, or magnetic resonance imaging.
Compounds
Agents of the present invention primarily activate the ostoeblastic PKC/Ca++
pathways. The pathway is shown in FIG. 1. Agents useful in the present
invention
increase bone volume by primarily activating the ostoeblastic PKC/Ca++
pathways.
Particularly preferred agents are agents that are selective for FP receptors.
Particularly
preferred agents selective for FP receptors are non-naturally-occurring FP
agonist.
Particularly preferred non-naturally-occurring FP agonists are selective for
the FP
receptor over other excitatory prostaglandin receptors in a ratio of at least
about 1:10,
more preferably at least about 1:20, and most preferably at least about 1:50.
Still more
preferred non-naturally-occurring FP agonists are selective for the FP
receptor over all
other prostanoid receptors in a ratio of at least about 1:10, more preferably
at least about
1:20, and most preferably at least about 1:50.
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Particularly useful non-naturally-occurring selective FP agonists are
prostaglandin analogs. Examples of such compounds are prostaglandin analogs
having
the following general structure:
~-~"~' R ~
HO
X Y
wherein:
R, is COZH, C(O)NHOH, COZRZ, CH,OH, S(O)zR2, C(O)NHRz, C(O)NHS(O)ZRZ,
or tetrazole; characterized in that Rz is alkyl, heteroalkyl, carbocyclic
aliphatic ring,
heterocyclic aliphatic ring, aromatic ring, or heteroaromatic ring;
X is (CH2)n, where n is 0 to 3, NH, S, or O; and
Y is a cycloalkyl or aromatic moiety, either substituted or unsubstituted.
Prostaglandin analogs of the above structure include: cloprostenol
(Estrumate~,
fluprostenol (Equimate~), tiaprost, alfaprostol, delprostenate, froxiprost,
latanoprost,
13,14-dihydro-16-((3-trifluoromethyl)phenoxy)-16-tetranor prostaglandin Fla,
17-((3-
trifluoromethyl}phenyl)-17-trinor-prostaglandin F2a, 13,14-dihydro-18-thienyl-
18-dinor
prostaglandin F 1 a and their analogs.
Other prostaglandin analogs of the present invention include 9-alpha, I1-
alpha,
15-alpha-trihydroxy-16-(3-chlorophenoxy)-omega-tetranor-prosta-4-cis-13-traps-
dienoic
acid and its analogs. Additional prostaglandin analogs are also disclosed in
CRC
Handbook of Eicosanoids: Prosta~landins and Related Lipids, Irolume l,
Chemical and
Biochemical Aspects, Part B. Ed. by Anthony L. Willis, CRC Press (Boca Raton,
1987)
Table Four pp. 80-97 (incorporated herein by reference}, and references
therein.
Methods of Use
The agents described above are useful in increasing bone volume, increasing
trabecular number through formation of new trabeculae, increasing bone mass
without
increasing the bone turnover rate, and increasing formation at the endosteal
surface
without removing bone from the existing cortex. Additionally, the quality of
bone
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formed by the administration of these agents is superior to that formed by the
administration of other bone anabolic agents, including prostaglandins of the
E series.
Bone quality refers to the combination of bone matrix (inorganic and organic),
bone
mass or volume, and bone architecture which impart overall strength and
fracture
resistance to bone. Accordingly, these agents are further useful in the
treatment and
prevention of a variety of bone disorders.
The preferred routes of administration for increasing bone volume and treating
bone disorders are transdermal and subcutaneous, e.g. injection or pellet.
Other preferred
routes of administration include oral, sublingual, and intranasal.
The dosage range for systemic administration of the non-naturally-occurnng FP
agonists of the present invention is from about 0.01 to about 1000 pg/kg body
weight per
day, preferably from about 0.05 to about 100 p.g/kg per body weight per day,
most
preferably from about 0.1 to about 50 pg/kg body weight per day. Plasma levels
are
expected to be in the range of about 0.01 to about 500 ng/ml, more preferably
from about
0.05 to 100 ng/ml, and most preferably from about 0.1 to 50 ng/ml.
While these dosages are based upon a daily administration rate, weekly or
monthly accumulated dosages may also be used to calculate the clinical
requirements.
The non-naturally-occurring FP agonists of the present invention may be
administered,
based on a weekly dosage, more frequently than once daily. The non-naturally-
occurring
FP agonists of the present invention may also be administered, based on a
weekly
dosage, less frequently than once daily. Hence, the weekly dosage may be
divided into
3, 4, 5, 6, or 7 daily dosages, preferably 5, 6, or 7 daily dosages.
Dosages may be varied based on the patient being treated, the condition being
treated, the severity of the condition being treated, and the route of
administration to
achieve the desired effect.
It has been further discovered that prolonged delivery (also referred to as
"prolonged administration") of the non-naturally-occurring FP agonist
unexpectedly
results in improved dose separation between side effects and the desired bone
effect.
That is, as used herein, "prolonged delivery" or "prolonged administration"
means that
the total daily dosage is delivered into the subject's circulation over a
period of at least
about 6 hours and up to 24 hours. Preferred prolonged delivery periods are for
at least
about 12 hours and up to 24 hours. Examples of prolonged delivery include
administration of the non-naturally-occurring FP agonist via a transdermal
patch or a
subcutaneous pump that delivers the total daily dosage over a twenty-four hour
period.
It is believed that the flattening of the plasma concentration curve resulting
from
prolonged delivery mitigates side effects while maintaining bone efficacy. It
is further
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believed that the administration of non-naturally-occurring FP agonists with
extended
half lives will likewise result in a flattening of the plasma concentration
curve without
prolonging the administration.
The following non-limiting examples serve to further illustrate the use of the
agents of the present invention.
Example I
The FP agonist, fluprostenol, is administered to a 65 year old woman who has
decreased bone mass and has been diagnosed with osteoporosis by her physician.
She is
treated daily with a transdermal patch that delivers 10 ug/kg fluprostenol
over a 24 hour
period. This treatment is continued for 24 months, at which time, vertebral
bone mass is
substantially increased compared to her vertebral bone mass at the onset of
therapy as
measured by dual energy X-ray absorptiometry (DXA).
Example II
The FP agonist, fluprostenol, is administered to a 63 year old woman who has
decreased bone mass and has been diagnosed with osteoporosis by her physician.
She is
treated with an implantable subcutaneous pump that delivers 10 pg/kg
fluprostenol over
a 24 hour period. This treatment is continued for 12 months, at which time,
vertebral
bone mass is substantially increased compared to her vertebral bone mass at
the onset of
therapy as measured by dual energy X-ray absorptiometry (DXA).
Pharmaceutical Formulations
Pharmaceutical formulations of the present invention comprise a safe and
effective amount of the non-naturally-occurring FP agonist and a
pharmaceutically
acceptable Garner.
The phrase "safe and effective amount", as used herein means an amount of a
compound or composition high enough to significantly positively modify the
symptoms
and/or condition to be treated, but low enough to avoid serious side effects
(at a
reasonable benefit/risk ratio), within the scope of sound medical judgment.
The safe and
effective amount of an agent for use in the method of the invention herein
will vary with
the particular condition being treated, the age and physical condition of the
patient being
treated, the severity of the condition, the duration of the treatment, the
nature of
concurrent therapy, the particular agent being employed, the particular
pharmaceutically-
acceptable excipients utilized, and like factors within the knowledge and
expertise of the
attending physician.
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In addition to the compound, the compositions of the subject invention contain
a
pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable
carrier", as
used herein, means one or more compatible solid or liquid filler diluents or
encapsulating
substances which are suitable for administration to a subject. The term
"compatible", as
used herein, means that the components of the composition are capable of being
commingled with the compound, and with each other, in a manner such that there
is no
interaction which would substantially reduce the pharmaceutical efficacy of
the
composition under ordinary use situations. Pharmaceutically-acceptable
carriers must, of
course, be of sufficiently high purity and sufficiently low toxicity to render
them suitable
for administration to the subject being treated.
Some examples of substances which can serve as pharmaceutically-acceptable
carriers or components thereof are sugars, such as lactose, glucose and
sucrose; starches,
such as cornstarch and potato starch; cellulose and its derivatives, such as
sodium
carboxymethyl cellulose, ethyl cellulose, cellulose acetate; powdered
tragacanth; malt;
gelatin; talc; solid lubricants, such as stearic acid, magnesium stearate;
calcium sulfate;
vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil,
corn oil and oil of
theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and
polyethylene glycol; alginic acid; emulsifiers, such as the Tweens~; wetting
agents such
as sodium lauryl sulfate; coloring agents; flavoring agents, excipients;
tableting agents;
stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline;
and
phosphate buffer solutions.
The choice of a pharmaceutically-acceptable carrier to be used in conjunction
with a compound is basically determined by the way the compound is to be
administered.
The non-naturally-occurring FP agonist of the present invention may be
administered
systemically, including transdermally, orally and/or parenterally, including
subcutaneous
or intravenous injection, and/or intranasally.
The appropriate amount of the agent, preferably non-naturally-occurring FP
agonist, to be used may be determined by routine experimentation with animal
models.
Such a model includes, but is not limited to, the intact and ovariectomized
rat models of
osteoporosis, the ferret, canine, and non human primate models of
osteoporosis, as well
as disuse models of osteoporosis.
A preferred method of administering non-naturally-occurring FP agonists is via
transdermal delivery. Preferred transdermal dosage forms include transdermal
patches,
creams, ointments, gels and the like. Another preferred method of
administering non-
naturally-occurring FP agonists is via subcutaneous injection in a unit dosage
form.
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Preferred unit dosage forms for injection include sterile solutions of water,
physiological
saline, or mixtures thereof. The pH of said solutions should be adjusted to
about 7.4.
Other preferred dose forms include nasal, rectal, sublingual, and oral.
Suitable
carriers for injection or surgical implants include hydrogels, controlled- or
sustained-
release devises, polylactic acid, and collagen matrices. Implant devices may
be coated
with the non-naturally-occurnng FP agonist. The non-naturally-occurring
prostaglandin
FP agonist may be dissolved in a buffer and may be mixed with a collagen gel
which is
then coated onto the porous end of the implant device.
Preferred oral forms include, for example liposomes, lipid emulsions,
proteinaceous cages and pharmaceutically-acceptable excipients.
The term "pharmaceutically-acceptable excipients" as used herein includes any
physiologically inert, pharmacologically inactive material known to one
skilled in the art,
which is compatible with the physical and chemical characteristics of the
particular
active ingredient selected for use. Pharmaceutically-acceptable excipients
include, but
are not limited to, polymers, resins, plasticizers, fillers, lubricants,
binders, disintegrants,
solvents, co-solvents, buffer systems, surfactants, preservatives, sweetening
agents,
flavoring agents, pharmaceutical grade dyes and pigments.
The following non-limiting examples illustrate formulations of the subject
invention.
Example III
Pharmaceutical formulations (compositions) in the form of tablets are prepared
by conventional methods, such as mixing and direct compaction, formulated as
follows
In redient Quantity (m~ per tablet,
Fluprostenol
Microcystalline Cellulose 100
Sodium Starch Glycollate 30
Magnesium Stearate 3
The above tablet administered orally once daily for six months substantially
increases bone volume of a patient afflicted with Osteoporosis.
Example IV
A pharmaceutical composition in liquid form is prepared by conventional
methods, formulated as follows:
In; edient uanti
Cloprostenol Smg
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Phosphate buffered physiologic saline 10 ml
Methyl paraben 0.05 ml
1.0 ml of the above composition administered subcutaneously once daily for six
months substantially increases bone volume of a patient afflicted with
osteoporosis.
While particular embodiments of the subject invention have been described, it
would be obvious to those skilled in the art that various changes and
modifications to the
compositions disclosed herein can be made without departing from the spirit
and scope
of the invention.
