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CA 02580281 2007-03-13
WO 2006/033912 PCT/US2005/032706
TREATMENT OF BONE DISORDERS WITH
SKELETAL ANABOLIC DRUGS
RELATED APPLICATIONS
This application is related to U.S.S.N. 10/340,484 filed January 10, 2003,
which is related
to U.S.S.N. 60/347,215, filed January 10, 2002; U.S.S.N. 60/353,296, filed
February 1, 2002;
U.S.S.N. 60/368,955, filed March 28, 2002; and U.S.S.N. 60/379,125, filed May
8, 2002; each of
which is hereby incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates generally to methods for the prevention and
treatment of a
variety of mammalian conditions manifested by loss of bone mass, including
osteoporosis. More
particularly, the present invention relates to methods of using PTHrP, or an
analog thereof, for
the treatment of metabolic bone disorders that are effective and have an
increased safety.
BACKGROUND OF THE INVENTION
Throughout adult life, bone continually undergoes remodeling through the
interactive
cycles of bone formation and resorption (bone turnover). Bone resorption is
typically rapid, and
is mediated by osteoclasts (bone resorbing cells), formed by mononuclear
phagocytic precursor
cells at bone remodeling sites. This process then is followed by the
appearance of osteoblasts
(bone forming cells), which form bone slowly to replace the lost bone. The
fact that completion
of this process normally leads to balanced replacement and renewal of bone
indicates that the
molecular signals and events that influence bone remodeling are tightly
controlled.
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 the
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 the
femoral bones and bones in the forearm and vertebrae predominantly. These
fractures, in turn,
lead to an increase to general morbidity, a marked loss of stature and
mobility, and in many
cases, an increase in mortality resulting from complications.
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A number of bone growth disorders are known which cause an imbalance in the
bone
remodeling cycle. Chief among these are metabolic bone diseases, such as
osteoporosis,
osteomalacia/rickets, chronic renal failure and hyperparathyroidism, which
result in abnormal or
excessive loss of bone mass (osteopenia).
Osteoporosis, or porous bone, is a disease characterized by low bone mass and
structural
deterioration of bone tissue, leading to bone fragility and an increased
susceptibility to fractures
of the hip, spine, and wrist. It is a devastating disease among both
postmenopausal women as
well as among older men. The costs at the national level for medications and
hospitalizations are
estimated to be in the $50,000,000 per year range at present and are likely to
increase as the US
population ages. At present, the mainstays of therapy are oral calcium
supplements, vitamin D
supplements, and a family of medications termed "anti-resorptives" which
reduce osteoclastic
bone resorption. These include estrogens, such as conjugated estrogens
(Premarin(D); selective
estrogen receptor modulators (SERMs), such as raloxifene (Evista ); calcitonin
(Miacalcin );
and bisphosphonates, such as alendronate (Fosamax ), risedronate (Actonel ),
etidronate
15- (Didronel ), pamidronate (Aredia ), tiludronate (Skelid ), or zoledronic
acid (Zometa ). See,
The writing group for the PEPI trial, JAMA 276: 1389-1396 (1996); Delmas et
al., NEngl JMed
337: ~ 1641-1647 (1997); Chestnut et al., Osteoporosis Int 8 (suppl 3): 13
(1998); Liberman et al.,
NEngl JMed 333: 1437-1443 (1995); McClung et al., NEngl JMed 344: 333-40
(2001). These
drugs are effective in slowing bone mineral loss and even cause moderate
increases in lumbar
spine bone mineral density in the range of 2% (calcium, vitamin D,
calcitonin), 3% (raloxifene),
6% (estrogens) or 8% (bisphosphonates). In general, two to three years of
administration are
required to achieve effects of this magnitude. See, The writing group for the
PEPI trial, JAMA
276: 1389-1396 (1996); Delmas et al., NEngl JMed 337: 1641-1647 (1997);
Chestnut et al.,
Osteoporosis Int 8(suppl3): 13 (1998); Liberman et al., NEngl JMed 333: 1437-
1443 (1995);
McClung et al., NEngl JMed 344: 333-40 (2001).
Osteoporosis exists, in general, when skeletal mineral losses are in the range
of 50%
below peak bone mass, which occurs at approximately age 30. Seen from the
perspective of
correcting the deficit in bone mineral, complete reversal of this 50% loss
would require a 100%
increase in bone mass. Thus, seen from this perspective, the 2-8% increases in
bone mineral
density which result from anti-resorptive therapy, while clinically
significant and beneficial,
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leaves very significant room for improvement. Since the use of anti-
resorptives to prevent bone
loss does not result in new bone production, the ultimate effectiveness of
anti-resorptives in
quantitative terms is limited. These considerations emphasize the need for the
development of
pharmaceutical mechanisms to produce new bone.
Recently, evidence has accumulated which clearly demonstrates that parathyroid
hormone (PTH) is a very effective new member of such a new osteoporosis
therapeutic
armamentarium. See, Finkelstein et aL, NEngl JMed 331: 1618-1623 (1994);
Hodsman et al., J
Clin Endocrinol Metab 82: 620-28 (1997); Lindsay et al., Lancet 350: 550-555
(1997); Neer et
al., N Engl JMed 344: 1434-1441 (2001); Roe et al., Program and Abstracts of
the 81 st Annual
Meeting of the Endocrine Society, p. 59 (1999); Lane et al., J Clin Invest
102: 1627-1633
(1998). PTH was first identified in parathyroid gland extracts in the 1920's.
The complete
amino acid sequence of PTH was determined in the 1970's. Because patients with
overproduction of parathyroid hormone (i.e., hyperparathyroidism) develop a
decline in bone
mass (sometimes very severe), PTH has widely been seen as a catabolic skeletal
agent over the
past century. However, both animal and human studies have now clearly
demonstrated that
when administered subcutaneously as a single daily dose, (so called
"intermittently" - in contrast
to the continuous overproduction of PTH which occurs in patients with
hyperparathyroidism),
PTH can induce marked increases in bone mineral density and bone mass. Thus,
PTH is very
different from the anti-resorptive class of drugs. See, Finkelstein et al.,
NEngl JMed 331:
1618-1623 (1994); Hodsman et al., J Clin Endocrinol Metab 82: 620-28 (1997);
Lindsay et al.,
Lancet 350: 550-555 (1997); Neer et al., NEngl JMed 344: 1434-1441 (2001); Roe
et al.d
Program and Abstracts of the 81 st Annual Meeting of the Endocrine Society, p.
59 (1999); Lane
et al., JClin Invest 102: 1627-1633 (1998). While the cellular basis for this
anabolic effect
remains to be defined, the effects at the microscopic and physiologic level
are clear: PTH when
administered intermittently results in an marked activation of bone-forming
osteoblasts, while
activating bone-resorbing osteoclasts to a lesser extent. These effects are
directionally opposite
from the anti-resorptive drugs described above, which inhibit both
osteoclastic and osteoblastic
activity.
To put these results in quantitative terms, PTH has been shown in multiple
studies to
increase lumbar spine bone mineral density by approximately 10-15%, depending
on the study
(see, Finkelstein et al., NEngl JMed 331: 1618-1623 (1994); Hodsman et al.,
JClin Endocrinol
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Metab 82: 620-28 (1997); Lindsay et al., Lancet 350: 550-555 (1997); Roe et
al., Program and
Abstracts of the 81 st Anniial Meeting of the Endocrine Society, p. 59 (1999);
Lane et al., J Clin
Invest 102: 1627-1633 (1998)). In one study, spine bone mineral density was
reported to be
increased by as much as 30%, when assessed using dual energy x-ray
absorptiometry (DXA),
and as much as 80% when using quantitative computerized tomography (QCT) of
lumbar spine
trabecular bone (see, Roe et al., Program and Abstracts of the 81 st Annual
Meeting of the
Endocrine Society, 59 (1999)).
In addition to increasing bone mass, PTH has recently been demonstrated to
have
significant anti-fracture efficacy, both at the spine and at non-vertebral
sites. PTH has been
shown to reduce fractures by between 60% and 90% depending on the skeletal
site and the
definition of fracture. Neer et al., NEngl JMed 344: 1434-1441 (2001). These
effects are at
least as pronounced as the anti-fracture efficacy of the anti-resorptives
(see, The writing group
for the PEPI trial, JAMA 276: 1389-1396 (1996); Delmas et al., NEngl JMed 337:
1641-1647
(1997); Chestnut et al., Osteoporosis Int 8(suppl 3): 13 (1998); Liberman et
al., NEngl JMed
333: 1437-1443 (1995); McClung et al., NEngl JMed 344: 333-40 (2001)), and may
be
superior. Thus, PTH appears to be the first member of a new class of anti-
osteoporosis drugs,
which in contrast to the anti-resorptives, have been termed the skeletal
"anabolic" class of
osteoporosis drugs, or "anabolics."
Parathyroid hormone-related protein (PTHrP) appears to be a second member of
this
class of skeletal anabolic drugs. See, Stewart et al., JBone Min Res 15: 1517-
1525 (2000).
PTHrP is the product of a gene distinct from that which encodes PTH. PTHrP
shares
approximately 60% homology at the amino acid level with PTH in the first 1.3
amino acids, and
then the sequences diverge completely. Yang et al., In: Bilezikian, Raisz, and
Rodan (Eds).
PRINCIPLES OF BONE BIOLOGY. Academic Press, San Diego CA, pp. 347-376 (1996).
PTHrP is
initially translated as a pro-hormone that'then undergoes extensive post-
translational processing.
One of the processed forms, or authentic secretory forms, as identified in the
inventor's
laboratory, is PTHrP-(1-36). Wu et al., JBiol Chem 271: 24371-24381 (1996).
PTHrP-(1-36)
binds to the common PTH/PTHrP receptor, also termed the PTH-1 receptor, in
bone and kidney.
Everhart-Caye et al., J Clin Endocrinol Metab, 81: 199-208 (1996); Orloff et
al., Endocrinology,
131: 1603-1611 (1992). PTHrP-(1-36) binds to this receptor with equal affinity
to PTH, and
activates the PKA and PKC signal transduction pathways with equal potency as
PTH. Everhart-
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Caye et al., J Clin Endocrinol Metab, 81: 199-208 (1996); Orloff et al.,
Endocrinology,131:
1603-1611 (1992).
PTHrP was originally identified by the inventor (Burtis et al., J Biol Chem
262:
7151-7156 (1987); Stewart et al., Biochem Biophys Res Comm 146: 672-678
(1987)) and others
(Strewler et al., J Clin Invest, 80: 1803, (1987); Moseley et al., Proc. Natl.
Acad. Sci. USA. 84:
5048-5052 (1987)) through its role as the causative agent for the common human
paraneoplastic
syndrome termed humoral hypercalcemia of malignancy (HHM). Stewart et al.,
NEngl JMed
303: 1377-1383 (1980). For example, humans with HH1Vl may lose as much as 50%
of their
skeletal mass over a period of a few months, as a result of sustained
elevations in circulating
PTHrP. Stewart et al., J Clin Endo Metab 55: 219-227 (1982). Subsequent animal
studies have
indicated that PTHrP is capable of increasing bone mass in osteoporotic rats
when administered
intermittently. Surprisingly, however, the increases in bone mineral density,
bone mass, bone
formation, and skeletal biomechanics induced by PTHrP were not as dramatic as
those observed
using equimolar quantities of PTH. Stewart et al., JBone Min Res 15: 1517-1525
(2000).
Nonetheless, there anabolic and biomechanic-enhancing effects of PTHrP are
surprising, since
PTHrP is widely viewed as the quintessential catabolic skeletal hormone
responsible for
dramatic skeletal mineral losses in patients with HHM. Stewart et al., J Clin
Endo Metab 55:
219-227 (1982). The observation that it is actually anabolic for the skeleton
when administered
intermittently was not anticipated, as evidenced by the fact that many
investigators and
pharmaceutical firms have worked for the past 10 years with PTH in
osteoporosis, but none has
embraced PTHrP despite its having been in the public domain since its initial
description in
1987.
In 1999, Eli Lilly released a report to the FDA that indicated that daily
administration of
PTH to rats over a two-year period resulted in the development of osteogenic
sarcomas in these
rats. See, FDA notification to PTH IND holders, December 11, 1998 (Neer et
al., NEngl JMed
344: 1434-1441 (2001)). The development of these malignant skeletal tumors is
extremely
troubling to experts in the field, because the development of skeletal tumors
derived from
osteoblasts in this preclinical toxicity model was biologically plausible in
causative terms, as
being related to PTH. One key concern in the rat osteosarcoma story is that
PTH was
administered in the preclinical toxicity studies to growing rats for two
years. This represents the
large majority of the lifespan of the rat, also approximately two years. In
humans, PTH
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treatment nas generaiiy naa a aurauon oi iwo to three years (Finkelstein et
al., NEngl JMed
331: 1618-1623 (1994); Hodsman et al., J Clin Endocrinol Metab 82: 620-28
(1997); Lindsay et
al., Lancet 350: 550-555 (1997); Neer et al., NEngl JMed 344: 1434-1441
(2001); Roe et al.,
Program and Abstracts of the 81 st Annual Meeting of the Endocrine Society, p.
59 (1999); Lane
et al., J Clin Invest 102: 1627-1633 (1998)). Most investigators anticipate
that the duration of
treatment with PTH will be from 18 months to 3 years. Therefore, a concern
remains in the
minds of some that long-term PTH treatment could result in osteosarcomas in
humans.
Accordingly, a need remains in the art for a method for the prevenlftsn and
treatment of
bone disorders using skeletal anabolic drugs that is both safe and effective.
SUMMARY OF THE INVENTION
The present invention provides methods for the prevention and treatment of a
variety of
mammalian conditions manifested by loss of bone mass, including osteoporosis.
The invention
is based on the surprising observation that the administration of very high
doses of a PTHrP, or a
related analog, can produce drastic increases in BMD in a very short time
period. The period of
administration is preferably 15, 18, 21, 24, 30, or 36 months, more preferably
7, 8, 9, 10, 11, or.
12 months, and most preferably 1, 2, 3, 4, 5, or 6 months. The high doses of
the skeletal
anabolic drug do not produce any significant adverse side effects when
administered for short
periods of time or at intermittant dosing intervals. Accordingly, the methods
of the present
invention offer greater safety by substantially eliminating or reducing the
risk of negative side
effects commonly associated with skeletal anabolic drugs, such as
hypercalcemia, renal failure,
hypotension, or the risk of developing osteogenic sarcomas.
The rates of increase in BMD achieved with the methods of the present
invention are
extremely rapid. In one embodiment, three months of treatment with PTHrP-(1-
36) yielded rates
of increase in BMD that were greater than any rates previously obtained with
anti-resorptives
and lower doses of PTH for longer periods of administrations. The rates of
increase in BMD
achieved with the methods of the present invention are preferably at least 1%
per month, 1.1%
per month or 1.2% per month, more preferably 1.3% per month or 1.4% per month,
and most
preferably over 1.5% per month or 1.6% per month.
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The increases in BMD observed are not generally obtained with anti-resorptives
for two
to three years of administrations. Indeed, several available anti-resorptives
(SERMs, calcitonin,
vitamin D, calcium) never achieve the increments in BMD obtained with the
methods of the
present invention. Moreover, the increments in BMD obtained with the methods
of the present
invention are comparable, or superior, to those achieved using lower doses of
PTH for longer
periods of administrations. Accordingly, the present invention provides
methods for the
prevention and treatment of bone disorders using skeletal anabolic drugs that
are both safe and
effective.
The resulting increase in BMD achieved with the methods of the present
invention
preferably results in T-scores >-2.5, more preferably results in T-scores >-
2.0, and most
preferably results in T-scores >-1Ø Furthermore, the resulting increase in
BMD achieved with
the methods of the present invention preferably prevents fractures resulting
preferably in at least
a 50%, 60%, or 70% reduction in incidence of fractures, more preferably in at
least a 75%; 80%,
or 85% reduction in incidence of fractures, and most preferably in at least a
90% or 95%
reduction in incidence of fractures.
In one aspect, the present invention provides methods of increasing bone mass
in an
animal or a human patient by administering intermittently to the patient
PTHrP, or an analog
thereof, at a dosage between 5 g/day and 50 mg/day or greater. A preferred
dose range is
10-45,000 g/day. Other preferred dose ranges include 25-40,000 g/day, 35-
37,500- g/day,
50-35,000 g/day, 100-30,000 g/day, 150-25,000 g/day, 200-20,000 g/day, 250-
15,000
g/day, 300-10,000 g/day, 350-7,500 g/day, 400-3,000 g/day, 400-1,500
g/day, 400-1,20G
g/day, 400-900 g/day, 400-600 g/day, 80-500 g/day, 90-500 g/day, 100-500
g/day,
150-500 g/day, 200-500 g/day, 250-500 g/day, 300-500 g/day, 350-500
g/day, 400-500
g/day, and 450-500 g/day. In a preferred embodiment, PTHrP-(1-36) is
administered at a
intermittant dosages between 50 and 10,000 g/day. A more preferred dose range
is 200-7,500
g/day. An even more preferred dose range is 400-5,000 g/day. Other preferred
dose ranges
include 400-1,500 g/day, 400-1,200 g/day, 400-900 g/day, and 400-600 g/day
(approximately 6.5-18 g/kg/day, 6.5-15 g/kg/day, 6.5-12 g/kg/day, and 6.5-9
g/kg/day).
The present invention also provides methods for increasing bone density using
administration of PTHrP, or analogs thereof, for periods of time longer than
previously
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administered in animals or humans. in one aspect, the present invention
provides methods of
increasing bone mass in an animal or a human patient by intermittently
administering PTHrP, or
an analog thereof, for a period of between 1-36 months. The period of
administration is
preferably 15, 18, 21, 24, 30, or 36 months, more preferably 7, 8, 9, 10, 11,
or 12 months, and
most preferably 1, 2, 3, 4, 5, or 6 months.
The methods of the invention can be employed with a patient afflicted with, or
at risk of,
a metabolic bone disorder including primary or secondary osteoporosis,
osteomalacia, renal
osteodystrophy, and other types of skeletal disorders with associated bone
loss. In one
embodiment, the rates of increase in B1VID achieved by the methods of the
present invention are
at least 1.5% per month. In another aspect, the PTHrP, or an analog thereof,
can be administered
to a patient afflicted with a bone fracture, e.g., a compound fracture or a
simple fracture.
Preferred embodiments include administration of PTHrP-(1-34) at the dosages
described above,
to patients afflicted with a bone fracture. In yet another aspect, the PTHrP,
or an analog thereof,
can be administered to a surgical patient to promote bone healing following
surgery, for example
hip replacement surgery or cardiac surgery, or other invasive procedures that
displace or damage
bone. Preferred embodiments of this include administration of PTHrP-(1-36) at
the dosages
described above. Other embodiments include PTHrP, fragments or analogs
thereof, administered
or applied to the damaged bone in formulations of bone pastes having the
dosages described
above.
PTHrP, or an analog thereof, used in the methods of the present invention can
be defined
by SEQ ID NO:2; have at least 70% homology with SEQ ID NO:2; or be encoded by
a nucleic
acid sequence that hybridizes under stringent conditions to a complementary
nucleic acid
sequence of SEQ ID NO:1. PTHrP analogs that can be used in the methods of this
invention
include fragments PTHrP-(1-30) through PTHrP-(1-173). PTHrP analogs can also
include
analogs with a model amphipathic alpha-helical peptide (MAP) sequence
substituted in the
C-terminal region of hPTHrP(1-34) such as [MAP1-10]22-31 hPTHrP-(1-34)NH2).
PTHrP
analogs can also include peptidomimetics and small molecule drugs having
skeletal anabolic
agonistic biological activities, as defined herein.
PTHrP can be administered by subcutaneous, oral, intravenous, intraperitoneal,
intramuscular, topically (on the bone surface e.g., as a paste or solution),
buccal, rectal, vaginal,
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intranasai and'aerosoi"admiriist"ation. 'Tif"e'rmittent administration may be
by periodic injections
once daily, once every two days, once every three days, once weekly, twice
weekly, biweekly,
twice monthly, and monthly. Alternatively, the use of pulsatile administration
of the skeletal
anabolic drug by mini-pump can be employed in the methods of the present
invention. Slow or
extended release matrices having PTHrP, fragments or analogs thereof, are also
suitable.
In yet another aspect, the present invention provides methods of increasing
bone mass in
an animal or a human patient. In one embodiment, the method comprises
administering between
1.5 and 90 mg of PTHrP, or an analog thereof, intermittently over a period
ranging from oine
week to one month. In another embodiment, the method comprises administering
between 3 and
180 mg of PTHrP, or an analog thereof, intermittently over a period ranging
from one week to
two months. In yet another embodiment, the method comprises administering
between 4.5 and
270 mg of PTHrP, or an analog thereof, intermittently over a period ranging
from one week to
three months. In yet another embodiment, the method comprises administering
between 9 and
540 mg of PTHrP, or an analog thereof, intermittently over a period ranging
from one week to
six months. In yet another embodiment, the method comprises administering
between 18 and
1080 mg of PTHrP, or an analog thereof, intermittently over a period ranging
from one week to
one year. In yet another embodiment, the method comprises administering
between 36 and 2160
mg of PTHrP, or an analog thereof, intermittently over a period ranging from
one week to two
years. In yet another embodiment, the method comprises administering between
54 and 3240 mg
of PTHrP, or an analog thereof, intermittently over a period ranging from one
week to three .
years. In still even another embodiment, the method comprises administering
between 70 and
10,000 mg of PTHrP, or an analog thereof, intermittently over a period ranging
from one week to
three years. In still even another embodiment, the method comprises
administering between 100
and 50,000 mg of PTHrP, or an analog thereof, intermittently over a
period'ranging from one
week to three years. According to these methods, the PTHrP, or analog thereof,
can be
administered at an intermittant dosing interval of twice daily, once daily,
once every two days,
once every three days, once-weekly, twice-weekly, biweekly, twice-monthly, or
monthly.
In yet another aspect, the present invention provides kit for increasing bone
mass in an
animal or a human patient. In one embodiment, the kit comprises between 1.5
and 90 mg of
PTHrP, or an analog thereof, and written directions providing instructions for
intermittent
administration of PTHrP, or an analog thereof, to an animal or a human patient
over a period
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ranging from one week to one month. In another embodiment, the kit comprises
between 3 and
180 mg of PTHrP, or an analog thereof, and written directions providing
instructions for
intermittent administration of PTHrP, or an analog thereof, to an animal or a
human patient over
a period ranging from one week to two months. In yet another embodiment, the
kit comprises
between 4.5 and 270 mg of PTHrP, or an analog thereof, and written directions
providing
instructions for intermittent administration of PTHrP, or an analog thereof,
to an animal or a
human patient over a period ranging from one week to three months. In yet
another
embodiment, the kit comprises between 9 and 540 mg of PTHrP, or an analog
thereof, and
written directions providing instructions for intermittent administration of
PTHrP, or an analog
thereof, to an animal or a human patient over a period ranging from one week
to six months. In
yet another embodiment, the kit comprises between 18 and 1080 mg of PTHrP, or
an analog
thereof, and written directions providing instructions for intermittent
administration of PTHrP, or
an analog thereof, to an animal or a human patient over a period ranging from
one week to one
year. In yet another embodiment, the kit comprises between 36 and 2160 mg of
PTHrP, or an
analog thereof, and written directions providing instructions for intermittent
administration of
PTHrP, or an analog thereof, to an animal or a human patient over a period
ranging from one
week to two years. In yet another embodiment, the kit comprises between 54 and
3240 mg of
PTHrP, or an analog thereof, and written directions providing instructions for
intermittent
administration of PTHrP, or an analog thereof, to an animal or a human patient
over a period
ranging from one week to three years. In still even another embodiment, the
kit comprises
between 70 and 10,000 mg of PTHrP, or an analog thereof, and written
directions providing
instructions for intermittent administration of PTHrP, or an analog thereof,
to an animal or a
human patient over a period ranging from one week to three years. In yet even
another
embodiment, the kit comprises between 100 and 50,000 mg of PTHrP, or an analog
thereof, and
written directions providing instructions for intermittent administration of
PTHrP, or an analog
thereof, to an animal or a human patient over a period ranging from one week
to three years.
The methods of the present invention can further comprise the step of co-
administering,
either simultaneously or sequentially with PTHrP, a bone resorption inhibiting
agent. The bone
resorption-inhibiting agent can be a bisphosphonate, estrogen, a selective
estrogen receptor
modulator, a selective androgen receptor modulator, calcitonin, a vitamin D
analog, or a calcium
salt. The bone resorption-inhibiting agent can also be alendronate,
risedronate, etidronate,
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õ . ..
pamidronate, tiludronate, zoledronic acid, raloxifene, tamoxifene,
droloxifene, toremifene,
idoxifene, levormeloxifene, or conjugated estrogens. In one embodiment, the
patient receives
intermittent administration of the skeletal anabolic drug for a three-month
period of time,
followed by a three-month period of treatment with a bone resorption-
inhibiting agent. A skilled
artisan will recognized that the sequential treatment regimen could begin with
a treatment period
with a bone resorption inhibiting agent followed by a treatment period with
the skeletal anabolic
drug, that the length of sequential treatment periods can be modified (e.g., 1-
18 months), and that
the skeletal anabolic drug can be co-administered with the bone resorption
inhibiting agent (e.g.,
sequential treatment period of a skeletal anabolic drug and a bone resorption
inhibiting agent
followed by a treatment period of a bone resorption inhibiting agent alone).
The sequential
treatment periods (e.g., three months of the skeletal anabolic drug followed
by three month of the
bone resorption inhibiting agent) can be repeated until the patient B1VID is
restored (e.g., a
T-score <-2.0 or -2.5 below the mean).
In still another aspect, the invention includes a computer system and methods
for the
design of peptidomimetics and small molecule drugs having skeletal anabolic
agonistic or
antagonistic biological activities. In one embodiment, the system includes a
processor, memory,
a display or data output means, a data input means, and a computer readable
instruction set
having at least an algorithm capable or rendering a three-dimensional
structure of a skeletal
anabolic agent, fragment, or derivative thereof, as well as a receptor for
such skeletal anabolic
agent. In a more preferred embodiment, the system comprises a computer aided
design (CAD)
algorithm capable of rendering a peptidomimetic or sma.ll molecule drug based
on the active sites
of the skeletal anabolic agent or receptor.
These and other objects of the present invention will be apparent from the
detailed
description of the invention provided below.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be further understood from the following
description with
reference to the tables, in which:
FIG. 1 is a homology alignment of human PTHrP-(1-36) with the corresponding
sequence in other species, aligned to maximize amino acid identity, and
wherein amino acids that
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differ from the correspondirig amin6 acid in the human sequence are bolded and
amino acids that
are conservative amino acid substitution variants of the corresponding amino
acids in the human
sequence are bolded and underlined.
FIG. 2 is a homology alignment of human PTH-(1=34) with the corresponding
sequence
in other species, aligned to maximize amino acid identity, and wherein amino
acids that differ
from the corresponding amino acid in the human sequence are bolded and amino
acids that are
conservative amino acid substitution variants of the corresponding amino acids
in the human
sequence are bolded and underlined.
FIG. 3 is a homology aligmnent of human TIP-(1-39) with the corresponding
sequence in
mouse, aligned to maximize amino acid identity, and wherein amino acids that
differ from the
corresponding amino acid in the human sequence are bolded and amino acids that
are
conservative amino acid substitution variants of the corresponding amino acids
in the human
sequence are bolded and underlined.
FIG. 4 is a line graph depicting the changes in lumbar vertebral bone mass
density
(BMD) expressed as % change (left panel) and weight (gram) change (right
panel) in
postmenopausal women with osteoporosis receiving placebo (N=7) or 410.25
g/day of
PTHrP-(1-36) (N=8).
FIG. 5 illustrates changes in bone mineral density as percent changes from
baseline,
following treatments with PTHrP or a placebo (PBO), as measured at the lumbar
spine (L/S), the
femoral neck (FN) and the total hip (TH). There is a marked increase in bone
mineral density at
the lumber spine in response to PTHrP treatment, a more moderate increase at
the femoral neck,
and approximately no increase in bone mineral density at the total hip.
FIG. 6 illustrates bone turnover markers in the placebo and PTHrP-treated
subjects. FIG.
6(a) demonstrates the serum osteocalcin results expressed as change from
baseline. FIG. 6(b)
indicates the serum N-telopeptide (NTX) values in the two groups. FIG. 6(c)
indicates the
urinary deoxypyridinolines in the two groups. The results demonstrate that
PTHrP stimulates
serum osteocalcin, and by inference, bone formation, but not bone resorption.
FIG. 7 illustrates serum total calcium (left panel) and ionized calcium (right
panel) in the
PTHrP and placebo groups. There is no difference in serum total or ionized
calcium between
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the, PTHrP and control groups, and no patient in either group developed
hypercalcemia as
measured by total or ionized serum calcium.
FIG. 8 is a line graph depicting the changes in lumbar vertebral bone mass
density
(BMD) expressed as % change in postmenopausal women with osteoporosis
receiving placebo
(N=7) or 410.25 g/day of PTHrP-(1-36) (N=8) compared to the effects of
various other
osteoporosis drugs reported in other published clinical studies.
FIG. 9 are line graphs depicting competition binding studies (Top Panels) of
121I_[Tyr36]PTHrP-(1-36)NH2 under equilibrium conditions to human renal
cortical membranes
(RCM) (Panel A), SaOS-2 membranes (Panel B), and SaOS-2 intact cells (Panel
C).
Competition curves are shown for unlabeled [Tyr36]PTHrP-(1-36)NH2 (,L), hPTH-
(1-34) (o),
rPTH-(1-34) (A), bPTH-(1-34) (0), [Tyr34]bPTH-(7-34)NHa (0), and hPTHrP-(7-
34)NH2 (-).
Values are the mean SEM of replicate determinations for a representative
experiment. Bottom
Panels are line graphs depicting the corresponding Scatchard transformations
of representative
binding experiments.
FIG. 10 are line graphs depicting the stimulation of adenylate cylcase
activity in human
renal cortical membranes (RCM) (Panel A), SaOS-2 membranes (Panel B), and SaOS-
2 intact
cells (Panel C) by [Tyr36]PTHrP-(1-36)NH2 (0), [Nleg 18,Tyr34]hPTH-(1-34)
(o),rPTH-(1-34)
(A), and bPTH-(1-34) (0). Assays were performed under the same conditions
employed in the
respective binding assays. Values are the mean SEM of replicate
determinations for a
representative experiment.
FIG 11 illustrates a line graph depicting the time course for binding of PTHrP
and PTH
peptides including 125I[Nle8 18, Tyr34]-hPTH-(1-34)NH2 to canine renal
membranes at 20 C:
--o-- total binding of radioligand; --~-- binding of radioligand in the
presence of 10"6M unlabeled
bPTH-(1-34) (nonspecific binding); --*-- specific binding of radioligand.
Points represent the
mean SEM of triplicate determinations. The SEM was too small, to indicate in
those points
without error bars. Results are representative of those obtained in three
experiments.
FIG. 12 is a line graph depicting competition binding studies of 125I-[Nle8
18, Tyr34]
hPTH-(1-34)NH2 to canine renal membranes at 20 C with unlabeled [Nle8 1$,
Tyr34]
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hPTHh-(1-34)NH2 (A), bPTH-(1-34) (0), and [Tyr36] PTHrP-(1-36)NH2 (o). Points
represent
the mean S.E. of triplicate determinations in three separate experiments
(bPTH-(1-34) and
[Tyr36]PTHrP-(1-36) amide) or in two separate experiments [Nle8'18, Tyr34]hPTH-
(1-34)NH2).
Individual points were expressed as a percentage of the specific binding
determined in the.
absence of unlabeled peptide (percentage of maximal binding). Inset indicates
Scatchard
analysis of a representative experiment. B/F, bound/free.
FIG. 13 is a line graph depicting competition binding studies of 121I-
[Tyr36]PTHrP-(1-36)
NH2 to canine renal membranes at 20 C with unlabeled [NleB 18,Tyr34]hPTH-(1-
34)NHz (A),
bPTH-(1-34) (*), [Tyr36]PTHrP-(l-36)NH2 (o), PTHrP-(49-74) (,L) and
[Cys5,Trp11,G1y13]
PTHrP-(5-18) (P1-PEPTIDE) (~). Points represent the mean S.E. of triplicate
determinations
in three separate experiments (bPTH-(1-34) and [Tyr36] PTHrP-(1-36) amide) or
in one
experiment [Nle8'18,Tyr34]hPTH-(1-34)NH2). Individual points were expressed as
a percentage
of the specific binding determined in the absence of unlabeled peptide
(percent of maximal
specific binding). Scatchard analysis (inset) of a representative experiment
is shown.
B/F, bound/free.
FIG. 14 illustrates the change in femoral bone mineral content in the five
groups. BMC
is shown as a percent change from the sham animals at each time point. Note
that there is a
progressive increase in femoral bone mineral content in each group of peptide-
treated rats, and
that the changes are highly significant in statistical terms.
FIG. 15 is a series of photomicrographs of the right proximal tibia following
90 days of
treatment. A. Sham; B. OVX; C. SDZ-PTH-893; D. rhPTH(1-34); E. hPTHrP(1-36).
Following
ovariectomy, bone is lost in the proximal tibia. Treatment with either SDZ-PTH-
893 or
PTH(1-34) for 90 days not only restores lost bone but significantly increases
trabecular bone
volume over Sham. PTHrP(1-36) only partially restores lost bone. Magnification
5.5x.
FIG. 16 depicts selected bone histomorphometric changes during the six month
period.
The key points are that: a) trabecular area, bone formation rate and
resorption surface decline
with age in the OVX groups; b) all three peptides had markedly positive
effects compared to
OVX controls on trabecular area and bone formation rate; and, c) despite this
marked increase in
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bone formation rate, bone resorption rates were similar in months 1-6 among
the treated and
control groups.
FIG. 17 illustrates changes in biomechanical strength (load to failure) during
the six
months of treatment. The key points are that:.a).marked.improvements in
biomechanical
measures occurred in all three groups for each of the three peptides; and b),
improvements
occurred at both predominantly trabecular and predominantly cortical sites.
FIG. 18 illustrates changes in serum calcium and renal calcium content during
the six
months. Note that rats treated with SDZ-PTH-893 developed moderate
hypercalcemia, and
marked increases in renal calcium content.
DETAILED DESCRIPTION OF THE INVENTION
A. GENERAL
Throughout adult life, bone is continually undergoing remodeling through the
interactive
cycles of bone formation and resorption (bone turnover). Bone resorption is
typically rapid, and
is mediated by osteoclasts (bone resorbing cells), formed by mononuclear
phagocytic precursor
cells at bone remodeling sites. This process then is followed by the
appearance of osteoblasts
(bone forming cells), which form bone slowly to replace the lost bone. The
activities of the
various cell types that participate in the remodeling process are controlled
by interacting
systemic (e.g., hormones, lymphokines, growth factors, vitamins) and local
factors (e.g.,
cytokines, adhesion molecules, lymphokines and growth factors). The fact that
completion of
this process normally leads to balanced replacement and renewal of bone
indicates that the
molecular signals and events that influence bone remodeling are tightly
controlled.
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 the
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 thefemoral bones and bones in the forearm and vertebrae
predominantly. These fractures, in turn,
lead to an increase to general morbidity, a marked loss of stature and
mobility, and in many
cases, an increase in mortality resulting from complications.
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A number of bone growth disorders are known which cause an imbalance in the
bone
remodeling cycle. Chief among these are metabolic bone diseases, such as
osteoporosis, rickets,
osteomalacia, chronic renal failure and hyperparathyroidism, which result in
abnormal or
excessive loss of bone mass (osteopenia). Other bone diseases, such as Paget's
disease, also
cause excessive loss of bone mass at localized sites.
Patients suffering from chronic renal (kidney) failure almost universally
suffer loss of
skeletal bone mass (renal osteodystrophy). While it is known that kidney
malfunction causes a
calcium and phosphate imbalance in the blood, to date replenishment of calcium
and phosphate
by dialysis does not significantly inhibit osteodystrophy in patients
suffering from chronic renal
failure. In adults, osteodystrophic symptoms often are a significant cause of
morbidity. In
children, renal failure often results in a failure to grow, due to the failure
to maintain and/or to
increase bone mass.
Rickets or Osteomalacia ("soft bones"), is a defect in bone mineralization
(e.g.,
incomplete mineralization), and classically is related to vitamin D (1,25-
dihydroxy vitamin D3)
deficiency or resistance. The defect can cause compression fractures in bone;
and a decrease in
bone mass, as well as extended zones of hypertrophy and proliferative
cartilage in place of bone
tissue. The deficiency may result from a nutritional deficiency (e.g., rickets
in children),
malabsorption of vitamin D or calcium, and/or impaired metabolism of the
vitamin.
Hyperparathyroidism (overproduction of the parathyroid hormone) has been known
to
cause abnormal bone loss since its initial description in the 1920's. In
children,
hyperparathyroidism can inhibit growth. In adults with hyperparathyroidism,
the skeleton
integrity is compromised and fractures of the hip, vertebrae, and other sites
are common. The
parathyroid hormone imbalance typically may result from parathyroid adenomas
or parathyroid
gland hyperplasia. Secondary hyperparathyroidism may result from a number of
disorders such
as vitamin D deficiency or prolonged pharmacological use of a glucocorticoid
such as cortisone.
Secondary hyperparathyroidism and renal osteodystrophy may result from chronic
renal failure.
In the early stages of the disease, osteoclasts are stimulated to resorb bone
in response to the
excess hormone present. As the disease progresses, the trabecular and cortical
bone may
ultimately be resorbed and marrow is replaced with fibrosis, macrophages, and
areas of
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hemorrhage as a consequence of microfractures. This condition, occurring in
both primary and
secondary hyperparathyroidism, is referred to pathologically as osteitis
fibrosa cystica.
Osteoporosis is a structural deterioration of the skeleton caused by loss of
bone mass
resulting from an imbalance in bone formation, bone resorption, or both, such
that the resorption
dominates the bone formation phase, thereby reducing the weight-bearing
capacity of the
affected bone. Osteoporosis affects >10 million individuals in the United
States, but only 10 to
20% are diagnosed and treated.
In a healthy adult, the rates at which bone is formed and resorbed are tightly
coordinated
so as to maintain the renewal of skeletal bone. However, in osteoporotic
individuals, an
imbalance in these bone-remodeling cycles develops which results in both loss
of bone mass and
in formation of microarchitectural defects in the continuity of the skeleton.
These skeletal
defects, created by perturbation in the remodeling sequence, accumulate and
finally reach a point
at which the structural integrity of the skeleton is severely compromised and
bone fracture is
likely. The chief clinical manifestations are vertebral and hip fractures, but
all parts of skeleton
may be affected. Osteoporosis is defined as a reduction-of bone mass (or
density) or the - -
presence of a fragility fracture. This reduction in bone tissue is accompanied
by deterioration in
the architecture of the skeleton, leading to a markedly increased risk of
fracture. Osteoporosis is
defmed operationally by the National Osteoporosis Foundation and World Health
Organization
as a bone density that falls -2.0 or -2.5 standard deviations (SD) below the
mean (also referred
to as a T-score of -2.0 or -2.5). Those who fall at the lower end of the young
normal range (a
T-score of > 1 SD below the mean) have low bone density and are considered to
be "osteopenic"
and be at increased risk of osteoporosis.
Although this imbalance occurs gradually in most. individuals..asthe.y age.
("s.enile ._.
osteoporosis"), it is much more severe and occurs at a rapid rate in
postmenopausal women. In
addition, osteoporosis also may result from nutritional and endocrine
imbalances, hereditary
disorders and a number of malignant transformations.
Epidemiology
In the United States, as many as 8 million women and 2 million men have
osteoporosis
(T-score <-2.5), and an additional 18 million individuals have bone mass
levels that put them at
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increased risk of developing osteoporosis (e.g., bone mass T-score <-1.0).
Osteoporosis occurs
more frequently with increasing age, as bone tissue is progressively lost. In
women, the loss of
ovarian function at menopause (typically after age 50) precipitates rapid bone
loss such that most
women meet the criteria for osteoporosis by age 70.
The epidemiology of fractures follows similar trends as the loss of bone
density.
Fractures of the distal radius increase in frequency before age 50 and plateau
by age 60, with
only a modest age-related increase thereafter. In contrast, incidence rates
for hip fractures
double every five years after age 70. This distinct epidemiology may be
related to the way
people fall as they age, with fewer falls on an outstretched hand. At least
1.5 million fractures
occur each year in the United States as a consequence of osteoporosis. As the
population
continues to age, the total number of fractures will continue to escalate.
Pathophysiology
Osteoporosis results from bone loss due to normal age-related changes in bone
remodeling as well as extrinsic and intrinsic factors that exaggerate this
process. These changes
may be superimposed on a low peak bone mass. Consequently, the bone remodeling
process is
fundamental for understanding the pathophysiology of osteoporosis. The
skeleton increases in
size by linear growth and by apposition of new bone tissue on the outer
surfaces of the cortex.
This latter process is the phenomenon of remodeling, which also allows the
long bones to adapt
in shape to the stresses placed upon them. Increased sex hormone production at
puberty is ,
required for maximum skeletal maturation, which reaches maximum mass and
density in early
adulthood. Nutrition and lifestyle also play an important role in growth,
though genetic factors
are the major determinants of peak skeletal mass and density. Numerous genes
control skeletal
growth, peak bone mass, and body size, but it is likely that separate genes
control skeletal
structure and density. Heritability estimates of 50 to 80% for bone density
and size have been
derived based on twin studies. Though peak bone mass is often lower among
individuals with a
family history of osteoporosis, association studies of candidate genes
[vitamin D receptor; Type I
collagen, the estrogen receptor (ER), interleukin (IL) 6; and insulin-like
growth factor (IGF) I]
have not been consistently replicated. Linkage studies suggest that several
genetic loci are
associated with high bone mass.
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Once peak skeletal mass has been attained, the process of remodeling remains
the
principal metabolic activity of the skeleton. This process has three primary
functions: (1) to
repair microdamage within the skeleton, (2) to maintain skeletal strength, and
(3) to supply
calcium from the skeleton to maintain serum calcium. Acute demands for calcium
involve
osteoclast-mediated resorption as well as calcium transport by osteocytes. The
activation of
remodeling may be induced by microdamage to bone due to excessive or
accumulated stress.
Bone remodeling is also regulated by several circulating hormones, including
estrogens,
androgens, vitamin D, and PTH, as well as locally produced growth factors such
as IGF-I and -II,
transforming growth factor (TGF) (3, PTHrP, ILs, prostaglandins, tumor
necrosis factor (TNF),
and osteoprotegrin and many others. Additional influences include nutrition
(particularly
calcium intake) and physical activity level. The end result of this remodeling
process is that the
resorbed bone is replaced by an equal amount of new bone tissue. Thus, the
mass of the skeleton
remains constant after peak bone mass is achieved in adulthood. After age 30
to 45, however,
the resorption and formation processes become imbalanced, and resorption
exceeds formation.
-15 This imbalance may begin at different ages and varies at different
skeletal sites; it becomes
exaggerated in women after menopause. Excessive bone loss can be due to an
increase in
osteoclastic activity and/or a decrease in osteoblastic activity. In addition,
an increase in
remodeling activation frequency can magnify the small imbalance seen at each
remodeling unit.
'Measurement of Bone Mass
Several noninvasive techniques are now available for estimating skeletal mass
or density.
These include dual-energy x-ray absorptiometry (DXA), single-energy x-ray
absorptiometry
(SXA), quantitative computed tomography (CT), and ultrasound.
DXA is a highly accurate x-ray technique that has become the standard for
measuring
bone density in most centers. Though it can be used for measurements of any
skeletal site,
clinical determinations are usually made of the lumbar spine and hip. Portable
DXA machines
have been developed that measure the heel (calcaneus), forearm (radius and
ulna), or finger
(phalanges), and DXA can also be used to measure body composition. In the DXA
technique,
two x-ray energies are used to estimate the area of mineralized tissue, and
the mineral content is
divided by the area, which partially corrects for body size. However, this
correction is only
partial since DXA is a two-dimensional scanning technique and cannot estimate
the depths or
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posteroanterior length of the bone. Thus, small people tend to have lower-than-
average bone
mineral density (BMD). Newer DXA techniques that measure infonnation B1VID are
currently
under evaluation. Bone spurs, which are frequent in osteoarthritis, tend to
falsely increase bone
density of the spine. Because DXA instrumentation is provided by several
different
manufacturers, the output varies in absolute terms. Consequently, it has
become standard
practice to relate the results to "normal" values using T-scores, which
compare individual results
to those in a young population that is matched for race and gender.
Alternatively, Z-scores
compare individual results to those of an age-matched population that is also
matched for race
and gender. Thus, a 60-year-old woman with a Z-score of-1 (1 SD below mean for
age) could
have a T-score of -2.5 (2.5 SD below mean for a young control group).
CT is used primarily to measure the spine, and peripheral CT is used to
measure bone in
the forearm or tibia. Research into the use of CT for measurement of the hip
is ongoing. CT has
the added advantage of studying bone density in subtypes of bone, e.g.,
trabecular vs. cortical.
The results obtained from CT are different from all others currently available
since this technique
specifically analyzes trabecular bone and can provide a true density (mass of
bone per unit
volume) measurement. However, CT remains expensive, involves greater radiation
exposure,
and is less reproducible.
Ultrasound is used to measure bone mass by calculating the attenuation of the
signal as it
passes through bone or the speed with which it traverses the bone. It is
unclear whether
ultrasound assesses bone quality, but this may be an advantage of the
technique. Because o.f its
relatively low cost and mobility, ultrasound is amenable for use as a
screening procedure.
All of these techniques for measuring BMD have been approved by the U.S. Food
and
Drug Administration (FDA) based upon their capacity to predict fracture risk.
The hip is the
preferred site of measurement in most individuals, since it directly assesses
bone mass at an
important fracture site. When hip measurements are performed by DXA, the spine
can be
measured at the same time. In younger individuals, such as perimenopausal
women, spine
measurements may be the most sensitive indicator of bone loss.
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B. STRUCTURAL AND t'UNCTIONAL PROPERTIES OF PTHRP PEPTIDES.
Parathyroid hormone-related peptide (PTHrP), a 140+ amino acid protein, and
fragments
thereof, reproduce the major biological actions of PTH. PTHrP is elaborated by
a number of
human and animal tumors and other tissues and may play a role in hypercalcemia
of 'malignancy.
The nucleotide and amino acid sequences of hPTHrP-(1-36) are provided in SEQ
ID NOS:1 and
2, respectively.
Biological activity is associated with the N-terminal portion. The amino acid
sequence of
the N-terminal segment of human PTHrP (hPTHrP) shows great homology with the N-
terminal
segment of various species, as illustrated in FIG. 1.
PTH and PTHrP, although distinctive products of different genes, exhibit
considerable
functional and structural homology and may have evolved from a shared
ancestral gene. The
structure of the gene for human PTHrP, however, is more complex than that of
PTH, containing
multiple exons and multiple sites for alternate splicing patterns during
formation of the mRNA.
Protein products of 141, 139, and 173 amino acids are produced, and other
molecular forms may
result from tissue-specific cleavage at accessible internal cleavage'sites.
The biologic roles of
these various molecular species and the nature of the circulating forms of
PTHrP are unclear. It
is uncertain whether PTHrP circulates at any significant level in normal human
adults; as a
paracrine factor, PTHrP may be produced, act, and be destroyed locally within
tissues. In adults
PTHrP appears to have little influence on calcium homeostasis, except in
disease states, when
large tumors, especially of the squamous cell type, lead to massive
overproduction of the
hormone.
The sequence homology between hPTH and hPTHrP is largely limited to the 13
N-terminal residues, 8 of which are identic.a.l.;.on1~.l.af 10._amino acids
in.the_(25..-34) receptor-
binding region of hPTH is conserved in hPTHrP. Conformational similarity may
underlie the
common activity. Cohen et al. (J. Bzol. Clhem. 266: 1997-2004 (1991)) have
suggested that
much of the sequence of PTH-(1-34) and PTHrP-(l -34), in particular regions (5-
18) and (21-34),
assumes an a-helical configuration, while noting that there is some question
whether this
configuration prevails for the carboxyl terminal end under physiological
conditions. Such a
secondary structure may be important for lipid interaction,=receptor
interaction, and/or structural
stabilization.
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The term "parathyroid hormone related protein" (PTHrP) encompasses naturally-
occurring PTHrP, as well as synthetic or recombinant PTHrP (rec PTHrP).
Further, the term
"parathyroid hormone related protein" encompasses allelic variants, species
variants, and
conservative amino acid substitution variants. The term also encompasses full-
length PTHrP-
(1-36), as well as PTHrP fragments, including small peptidomimetic molecules
having PTHrP-
like bioactivity, for example, in the assays described herein. As with PTH,
the biological activity
of PTHrP is associated with the N-terminal portion, with residues (1-30)
apparently the
minimum required. It will thus be understood that fragments of PTHrP variants,
in amounts
giving equivalent biological activity to PTHrP-(1-36), can be used in the
methods of the
invention, if desired. Fragments of PTHrP incorporate at least the amino acid
residues of PTHrP
necessary for a biological activity similar to that of intact PTHrP-(1-36).
Examples of such
fragments include PTHrP-(1-30), PTHrP-(1-31), PTHrP-(1-32), PTHrP-(1-33),
PTHrP-(1-34),
PTHrP-(l-35), PTHrP-(1-36), ... PTHrP-(1-139), PTHrP-(1-140), and PTHrP-(1-
141).
The term "parathyroid hormone-related protein" also encompasses variants and
functional
analogues of PTHrP having an homologous amino acid sequence with PTHrP-(1-36).
The
present invention thus includes pharmaceutical formulations comprising such
PTHrP variants
and functional analogs, carrying modifications like substitutions, deletions,
insertions, inversions
or cyclisations, but nevertheless having substantially the biological
activities of parathyroid
hormone. According to the present invention, "homologous amino acid sequence"
means an
amino acid sequence that differs from an amino acid sequence shown in SEQ ID
NO:2, by one or
more conservative amino acid substitutions, or by one or more non-conservative
amino acid
substitutions, deletions, or additions located at positions at which they do
not destroy the
biological activities of the polypeptide. Conservative amirio acid
substitutions typically include
substitutions among amino acids of the same class. These classes include, for
example, (a)
amino acids having uncharged polar side chains, such as asparagine, glutamine,
serine,
threonine, and tyrosine; (b) amino acids having basic side chains, such as
lysine, arginine, and
histidine; (c) amino acids having acidic side chains, such as aspartic acid
and glutamic acid; and
(d) amino acids having nonpolar side chains, such as glycine, alanine, valine,
leucine, isoleucine,
proline, phenylalanine, methionine; tryptophan, and cysteine. Preferably, such
a sequence is at
least 75%, preferably 80%, more preferably 85%, more preferably 90%, and most
preferably
95% homologous to the amino acid sequence in SEQ ID NO:2.
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According to the present invention, homologous amino acid sequences include
sequences
that are identical or substantially identical to an amino acid sequence as
shown in SEQ ID NO:2.
By "amino acid sequence substantially identical" is meant a sequence that is
at least 60%,
preferably 70%, more preferably 80%, more preferably 90%, and most preferably
95% identical
to an amino acid sequence of reference. Preferably the homologous sequence
differs from the
reference sequence, if at all, by a majority of conservative amino acid
substitutions.
The calculation of % homology and % identity are determined by first aligning
a
candidate PTHrP polypeptide with SEQ ID NO:2, as provided in FIG. 1. Once
aligned, the total
number of identical amino acids and/or the number of conservative amino acid
substitution
variants shared between the candidate polypeptide and SEQ ID NO:2 are counted.
For the
calculation of % identity, the number of identical amino acids between the
candidate PTHrP
polypeptide and the reference sequence is divided by the total number of amino
acids in the
reference sequence, and this number is multiplied by 100 to obtain a
percentage value. For the
calculation of % homology, the total number of identical amino acids and
conservative amino
acid substitution variants between the candidate PTHrP polypeptide and the
reference sequence
is divided by the total number of amino acids in the reference sequence, and
is multiplied by 100
to obtain a percentage value. FIG. 1 provides a homology alignment of human
PTHrP-(1-36)
(SEQ ID NO:2) with the corresponding sequence in other species, aligned to
maximize amino
acid identity. The amino acids in other species that differ from the
corresponding amino acid in
the human sequence are bolded and amino acids that are conservative amino acid
substitution
variants of the corresponding amino acids in the human sequence are bolded and
underlined.
The values of % identity and % homology are provided.
Alternatively, homology can be measured using sequence analysis software
(e.g.,
Sequence Analysis Software Package of the Genetics Computer Group, University
of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar
amino acid
sequences are aligned to obtain the maximum degree of homology (i.e.,
identity). To this end, it
may be necessary to artificially introduce gaps into the sequence. Once the
optimal alignment
has been set up, the degree of homology (i.e., identity) is established by
recording all of the
positions in which the amino acids of both sequences are identical, relative
to the total number of
positions.
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Similarity factors include similar size, shape and electrical charge. One
particularly
preferred method of determining amino acid similarities is the PAM25O matrix
described in
Dayhoff et aL, 5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE 345-352 (1978 &
Suppl.),
incorporated by reference herein. A similarity score is first calculated as
the sum of the aligned
pairwise amino acid similarity scores. Insertions and deletions are ignored
for the purposes of
percent homology and identity. Accordingly, gap penalties are not used in this
calculation. The
raw score is then normalized by dividing it by the geometric mean of the
scores of the candidate
compound and the reference sequence. The geometric mean is the square root of
the product of
these scores. The normalized raw score is the percent homology.
Polypeptides having a sequence homologous to one of the sequences shown in SEQ
ID
NOS:l or 2, include naturally-occurring allelic variants, as well as mutants
and variants or any
other non-naturally-occurring variants that are analogous in terms of bone
formation activity, to a
polypeptide having a sequence as shown in SEQ ID NO:2.
An allelic variant is an alternate form of a polypeptide that is characterized
as having a
substitution, deletion, or addition of one or more amino acids that does not
substantially alter the
biological function of the polypeptide. By "biological function" is meant the
function of the
polypeptide in the cells in which it naturally occurs, even if the function is
not necessary for the
growth or survival of the cells. For example, the biological function of a
porin is to allow the
entry into cells of compounds present in the extracellular medium. A
polypeptide can have more
than one biological function.
Allelic variants are very common in nature. Allelic variation may be equally
reflected at
the polynucleotide level. Polynucleotides, e.g., DNA molecules, encoding
allelic variants can
easily be retrieved by polymerase chain reaction (PCR) amplification of
genomic DNA extracted
by conventional methods. This involves the use of synthetic oligonucleotide
primers matching
upstream and downstream of the 5' and 3' ends of the encoding domain. Suitable
primers can be
designed according to the nucleotide sequence information provided in SEQ ID
NO: 1.
Typically, a primer can consist of 10-40, preferably 15-25 nucleotides. It may
be also
advantageous to select primers containing C and G nucleotides in a proportion
sufficient to
ensure efficient hybridization; e.g., an amount of C and G nucleotides of at
least 40%, preferably
50% of the total nucleotide amount.
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Useful homologs that do not naturally occur can be designed using known
methods for
identifying regions of a PTHrP peptide that are likely to be tolerant of amino
acid sequence
changes and/or deletions. For example, stability-enhanced or modified variants
of PTHrP are
known in the art. For example, Vickery et al, (J. Bone Miner. Res., 11: 1943-
1951 (1996))
described a PTHrP analog with a model amphipathic alpha-helical peptide (MAP)
sequence
substituted in the C-terminal region of hPTHrP(1-34) and reported that the
resulting analog,
[MAPl-10]22-31 hPTHrP-(1-34)NH2), had greater anabolic activity than the
parent peptide in
ovariectomized osteopenic rats. Other biologically active synthetic
polypeptide analogs of PTH
and PTHrP have been described in which amino acid residues (22-31) are
substituted with
hydrophilic amino acids and lipophilic amino acids forming an amphipathic a-
helix. See, e.g.,
U.S. Patent Nos: 5,589,452; 5,693,616; 5,695,955; 5,798,225; 5,807,823;
5,821,225; 5,840,837;
5,874,086; and 6,051,686, each of which is incorporated herein by reference.
These homologs
and other such biologically active peptidomimetic compounds are useful for
creating small-
molecule agonists or antagonists of PTHrP, PTH, or TIP peptides, as is
discussed in Example 6.
Polypeptide derivatives that are encoded by polynucleotides of the invention
include,
e.g., fragments, polypeptides having large internal deletions derived from
full-length
polypeptides, and fusion proteins.
Polypeptide fragments of the invention can be derived from a polypeptide
having a
sequence homologous to any of the sequences shown in SEQ ID NOS:2-13, to the
extent that the
fragments retain the desired substantial bone formation properties of the
parent polypeptide. '
A polynucleotide of the invention, having a homologous coding sequence, can
hybridize,
preferably under stringent conditions, to a polynucleotide having a sequence
complementary to
the nucleotide sequence in SEQ ID NO:1. Hybridization procedures are described
in, e.g.,
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons Inc.
(1994);
Silhavy et al., EXPERIMENTS WITH GENE FUSIONS, Cold Spring Harbor Laboratory
Press (1984);
Davis et al., A MANUAL FOR GENETIC ENGINEERING: ADVANCED BACTERIAL GENETICS,
Cold
Spring Harbor Laboratory Press (1980), each incorporated herein by reference.
Important
parameters that can be considered for optimizing hybridization conditions are
reflected in a
formula that allows calculation of a critical value, the melting temperature
above which two
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complementary DNA strands separate from each other. Lasey ana liaviason,
lvuci. a.cia lces. 4:
1539 (1977). This formula is as follows:
Tm = 81.5 + 0.5 x (% G+C) + 1.6 log (positive ion concentration) - 0.6 x (%
formamide).
Under appropriate stringency conditions, hybridization temperature (Th) is
approximately
20-40 C, 20-25 C or, preferably, 30-40 C below the calculated Tm. Those
skilled in the art will
understand that optimal temperature and salt conditions can be readily
determined empirically in
preliminary experiments using conventional procedures.
For example, stringent conditions can be achieved, both for pre-hybridizing
and
hybridizing incubations, (i) within 4-16 hours at 42 C, in 6xSSC containing
50% formamide or
(ii) within 4-16 hours at 65 C in an aqueous 6xSSC solution (1 M NaCl, 0.1 M
sodium citrate
(pH 7.0)).
For polynucleotides containing 30 to 600 nucleotides, the above formula is
used and then
is corrected by subtracting (600/polynucleotide size in base pairs).
Stringency conditions are
defined by a Th that is 5 to 10 C below Tm.
Hybridization conditions with oligonucleotides shorter than 20-30 bases do not
exactly
follow the rules set forth above. In such cases, the formula for calculating
the Tm is as follows:
Tm = 4 x (G+C) + 2 (A+T).
For example, an 18 nucleotide fragment of 50% G+C would have an approximate Tm
of 54 C.
Consequently, the methods of the present invention includes the use of a PTHrP
peptide
selected from the group consisting of:
(a) full-length PTHrP;
(b) biologically-active variants of full-length PTHrP;
(c) biologically active PTHrP fragments;
(d) biologically active variants of PTHrP fragments;
(e) biologically active variants having at least 75% homology with SEQ ID
NO:2;
(f) biologically active variants having at least 60% identity with SEQ ID
NO:2; and
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(g) biologically active variants encoded by a nucleic acid sequence that,
hybridizes
under stringent conditions to a complementary nucleic acid sequence of SEQ ID
NO:1.
PTHrP includes, but is not limited to, human PTHrP (hPTHrP), bovine PTHrP
(bPTHrP),
and rat PTHrP (rPTHrP). An analog of PTHrP is a peptide which is a structural
analog or
fragment (preferably, an N-terminal fragment containing 50 or fewer amino
acids) of a naturally-
occurring PTHrP and, like PTHrP, also capable of binding to PTH receptor and
stimulating
adenylate cyclase activity, thereby promoting bone formation. Examples of such
fragments
include, but are not limited to, PTHrP-(1-30), PTHrP-(1 -3 1), PTHrP-(1-32),
PTHrP-(1-33),
PTHrP-(l -34), PTHrP-(1-35), PTHrP-(1-36), ... PTHrP-(1-139), PTHrP-(1-140),
and PTHrP-
(1-141). The following publications disclose the sequences of PTHrP peptides:
Yasuda et al., J.
Biol. Chem. 264: 7720-7725 (1989); Schermer, J. Bone & Min. Res. 6: 149-155
(1991); and
Burtis, Clin. Chem. 38: 2171-2183 (1992). More examples can be found in the
following
publications: German Application 4203040 Al (1993); PCT Application 94/01460
(1994); PCT
Application 94/02510 (1994); EP Application 477885 A2(1992); EP Application
561412 Al
(1993); PCT Application 93/20203 (1993); U.S. Pat. No. 4,771,124 (1988); PCT
Application
92/11286 (1992); PCT Application 93/06846 (1993); PCT Application 92/10515
(1992); U.S.
Pat. No. 4,656,250 (1987); EP Application 293158 A2 (1988); PCT Application
94/03201
(1994); EP Application 451,867 Al (1991); U.S. Pat. No. 5,229,489 (1993); and
PCT
Application 92/00753 (1992).
PTHrP exerts important developmental influences on fetal bone development and
in adult
physiology. A homozygous knockout of the PTHrP gene (or the gene for the PTH
receptor) in
mice causes a lethal deformity in which animals are born with severe skeletal
deformities
resembling chondrodysplasia.
Many different cell types produce PTHrP, including brain, pancreas, heart,
lung,
mammary tissue, placenta, eridothelial cells, and smooth muscle. In fetal
animals, PTHrP directs
transplacental calcium transfer, and high concentrations of PTHrP are produced
in mammary
tissue and secreted into milk. Human and bovine milk, for example; contain
very high
concentrations of the hormone; the biologic significance of the latter is
unknown. PTHrP may
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also play a role in uterine contraction and other biologic functions, still
being clarified in other
tissue sites.
PTHRP Biological Actions
Because PTHrP shares a significant homology with PTH in the critical amino
terminus, it
binds to and activates the PTH/PTHrP receptor, with effects very similar to
those seen with PTH.
However, PTHrP, not PTH, appears to be the predominant physiologic regulator
of bone mass,
with PTHrP being essential for the development of full bone mass.
Demonstrating this,
conditional gene knockout strategies, employing mice in which the PTHrP gene
was disrupted in
osteoblasts prevented the production of PTHrP locally within adult bone, but
which had normal
PTH levels in adult bone. Absent PTHrP, and these mice developed osteoporosis
demonstrating
that osteoblast-derived PTHrP exerts anabolic effects in bone by promoting
osteoblast function:
See, Karaplis, A.C. "Conditional Knockout of PTHrP in Osteoblasts Leads to
Premature
Osteoporosis." Abstract 1052, Annual Meeting of the American Society for Bone
and Mineral
Research, September 2002, San Antonio, TX. JBone Mineral Res, Vol 17 (Suppl
1), pp S138,
2002, incorporated by reference. These findings indicate that PTHrP, and not
PTH, is the more
important normal regulator of bone mass under normal physiologic conditions,
and that PTH
treatment for osteoporosis, while effective, serves only as a surrogate for
PTHrP, the authentic
bone mass regulator.
The 500-amino-acid PTH/PTHrP receptor (also known as the PTH1 receptor)
belongs to
a subfamily of GCPR that includes those for glucagon, secretin, and vasoactive
intestinal
peptide. The extracellular regions are involved in hormone binding, and the
intracellular
domains, after hormone activation, bind G protein subunits to transduce
hormone signaling into
cellular responses through stimulation of second messengers.
A second PTH receptor (PTH2 receptor) is expressed in brain, pancreas, and
several
other tissues. Its amino acid sequence and the pattern of its binding and
stimulatory response to
PTH and PTHrP differ from those of the PTH1 receptor. The PTH/PTHrP receptor
responds
equivalently to PTH and PTHrP, whereas the PTH2 receptor responds only to PTH.
The
endogenous ligand of this receptor appears to be tubular infundibular peptide-
39 or TIP-39. The
physiological significance of the PTH2 receptor-TIP-39 system remains to be
defined. Recently,
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a 39-amino-acid hypothalamic peptide, tubular infundibular peptide (TIP-39),
has been
characterized and is a likely natural ligand of the PTH2 receptor.
The PTH1 and PTH2 receptors can be traced backward in evolutionary time to
fish. The
zebrafish PTHl and PTH2 receptors exhibit the same selective responses to PTH
and PTHrP as
do the human PTH1 and PTH2 receptors. The evolutionary conservation of
structure and
function suggests unique biologic roles for these receptors.
G proteins of the GS class link the PTH/PTHrP receptor to adenylate cyclase,
an enzyme
that generates cyclic AMP, leading to activation of protein kinase A. Coupling
to G proteins of
the Gq class links hormone action to phospholipase C, an enzyme that generates
inositol
phosphates (e.g., IP3) and DAG, leading to activation of protein kinase C and
intracellular
calcium release. Studies using the cloned PTH/PTHrP receptor confirm that it
can be coupled to
more than one G protein and second-messenger kinase pathway, apparently
explaining the
multiplicity of pathways stimulated by PTH and PTHrP. Incompletely
characterized second-
inessenger responses (e.g., MAP kinase activation) may be independent of
phospholipase C or
adenylate cyclase stimulation (the latter, however, is the strongest and best
characterized second
messenger signaling pathway for PTH and PTHrP).
The details of the biochemical steps by which an increased intracellular
concentration of
cyclic AMP,1P3, DAG, and intracellular Caz+ lead to ultimate changes in ECF
calcium and
phosphate ion translocation or bone cell function are unknown. Stimulation of
protein kinases
(A and C) and intracellular calcium transport is associated with a variety of
hormone-specific
tissue responses. These responses include inhibition of phosphate and
bicarbonate transport,
stimulation of calcium transport, and activation of renal 1 a-hydroxylase in
the kidney. The
responses in bone include effects on collagen synthesis; increased alkaline
phosphatase, ornithine
decarboxylase, citrate decarboxylase, and glucose-6-phosphate dehydrogenase
activities; DNA,
protein, and phospholipid synthesis; calcium and phosphate transport; and
local cytokine/growth
factor release. Ultimately, these biochemical events lead to an integrated
hormonal response in
bone turnover and calcium homeostasis.
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C. OTHER ANABOLIC AGENTS
Other agents provide anabolic effects, similar to those demonstrated by PTHrP,
for
example, PTH, and TIP. Compositions of PTH and TIP, and their uses, are
similar to those for
PTHrP disclosed herein. These skeletal anabolic agents, PTH and TIP, or
analogs thereof,
increase bone mass in a human patient in need thereof,.when administered to
said patient at a
dosage between 10 and 3,000 g/day for a period of 1-36 months. In alternative
embodiments,
the dosage is preferably 10 and 50,000 g/day, 20 and 30,000 g/day, 35 and
20,000 g/day, 40
and 15,000 g/day, 45 and 10,000 g/day, 50-5,000 g/day, more preferably 75-
1,500 g/day,
even more preferably 100-1,200 g/day, and most preferably 300-1,000 g/day.
In yet other
alternative embodiments, the period of administration is preferably 12, 15, or
18 months, more
preferably 7, 8, 9, 10, or 11 months, and most preferably 1, 2, 3, 4, 5, or 6
months. The incre,ase
in bone mass can be monitored by the assays described herein. These skeletal
anabolic agents
can be combined with PTHrP. They are described below.
PTH Peptides
PTH is an 84 amino-acid single-chain peptide. The amino acid sequence of PTH
has
been characterized in multiple mammalian species, revealing marked
conservation in the amino-
terminal portion, which is critical for many biologic. actions of the
molec.ule.. _ Biological activity
is associated with the N-terminal portion, with residues (1-29) apparently the
minimum required.
The N-terminal segment of human PTH (hPTH) differs from the N-terminal segment
of the
bovine (bPTH) and porcine (pPTH) hormones by only three and two amino acid
residues,
respectively.
PTH is initially synthesized as a larger molecule (preproparathyroid hormone,
consisting
of 115 amino acids), which is then reduced in size by signal peptide cleavage
(proparathyroid
hormone, 90 amino acids) and then a second prohormone cleavage before
secretion as an 84
amino acid peptide. The hydrophobic regions of the preproparathyroid hormone
serve a role in
guiding transport of the polypeptide from sites of synthesis on polyribosomes
through the
endoplasmic reticulum to secretory granules.
Modified, substituted synthetic fragments of the amino-terminal sequence as
small as
1-14 residues are sufficient to activate the major receptor. Biologic roles
for the carboxyl-
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tenninal region of PTH (e.g., 35-84) are under investigation; a separate
receptor or receptors may
exist for this region of the molecule. Fragments shortened or modified at the
amino terminus
still bind to the PTH receptor but lose the capacity to stimulate biologic
responses. For example,
the peptide composed of the sequence 7-34 is a competitive inhibitor of active
hormone binding
to receptors in vitro but is a weak inhibitor in vivo.
The term "parathyroid hormone" (PTH) encompasses naturally occurring PTH, as
well as
synthetic or recombinant PTH (rec PTH). Further, the term "parathyroid
hormone" encompasses
allelic variants, species variants, and conservative amino acid substitution
variants. The term
also encompasses full-length PTH-(1-84), as well as PTH fragments. It will
thus be understood
that fragments of PTH variants, in amounts giving equivalent biological
activity to PTH-(1-84),
can be used in the methods of the invention, if desired. Fragments of PTH
incorporate at least
the amino acid residues of PTH necessary for a biological activity similar to
that of intact PTH.
Examples of such fragments include: PTH-(1-29), PTH-(1-30), PTH-(1-31), PTH-(1-
32), PTH-
(1-33), PTH-(1-34), PTH-(1-80), PTH-(1-81), PTH-(1-82), PTH-(1-83), and PTH-(1-
84).
The term "parathyroid hormone" also encompasses variants and functional
analogs of
PTH having a homologous amino acid sequence with PTH-(l-34). The present
invention thus
includes pharmaceutical formulations comprising such PTH variants and
functional analogs,
carrying modifications like substitutions, deletions, insertions, inversions
or cyclisations, but
nevertheless having substantially the biological activities of parathyroid
hormone. Stability-
enhanced variants of PTH are known in the art from, e.g., WO 92/11286 and WO
93/20203, 'each
incorporated herein by reference. Variants of PTH can incorporate, for
example, amino acid
substitutions that improve PTH stability and half-life, such as the
replacement of methionine
residues at positions 8 and/or 18, and replacement of asparagine at position
16. Cyclized PTH
analogs are disclosed in, e.g., WO 98/05683, incorporated herein by reference.
The term
"parathyroid hormone" also encompasses amino acid substituted analogs using
the PTH-(1-11)
or PTH-(1-14) backbone. Shimizu et al., JBiol Chem., 276: 49003-49012 (2001);
Shimizu et
-al., Endocrinology 42: 3068-3074 (2001); Carter and Gardella, Biochim Biophys
Acta 1538:
290-304 (2001); Shimizu et al., JBiol Chem., 275: 21836-21843 (2000), each
incorporated
herein by reference.
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FIG. 2 provides a homology alignment of the reference sequence, human PTH-(1-
34)
(SEQ ID NO: 15), with the corresponding sequence in other species, aligned to
maxinlize amino
acid identity. "Homologous amino acid sequence" means an amino acid sequence
that differs
from an amino acid sequence shown in SEQ ID NO: 15, by one or more
conservative amino acid
substitutions, or by one or more non-conservative amino acid substitutions,
deletions, or
additions located at positions at which they do not destroy the biological
activities of the
polypeptide. Preferably, such a sequence is at least 75%, preferably 80%, more
preferably 85%,
more preferably 90%, and most preferably 95% homologous to the amino acid
sequence in SEQ
ID NO: 2. Homologous amino acid sequences also include sequences that are
identical or
substantially identical to an amino acid sequence as shown in SEQ ID NO: 15.
By "amino acid
sequence substantially identical" is meant a sequence that is at least 60%,
preferably 70%, more
preferably 80%, more preferably 90%, and-most preferably 95% identical to an
amino acid
sequence of reference. Preferably the homologous sequence differs from the
reference sequence,
if at all, by a majority of conservative amino acid substitutions.
PTH peptides useful in the methods of the present invention include the use of
a PTH
peptide selected from the group consisting of:
(a) full-length parathyroid hormone;
(b) biologically active variants of full-length parathyroid hormone;
(c) biologically active parathyroid hormone fragments;
(d) biologically active variants of parathyroid hormone fragments;
(e) biologically active variants having at least 75% homology with SEQ ID NO:
15;
(f) biologically active variants having at least 60% identity with SEQ ID
NO:15; and
(g) biologically active variants encoded by a nucleic acid sequence that
hybridizes
under stringent conditions to a complementary nucleic acid sequence of SEQ ID
NO:14.
TIP Peptides
Recently, a 39-amino-acid hypothalamic peptide, tubular infundibular peptide
(TIP-39),
has been characterized and is a likely natural ligand of the PTH2 receptor.
Accordingly, TIP-39,
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and biologically-active fragments and analogs thereof, can be used in the
methods of the present
invention.
The term "tubular infundibular peptide" encompasses naturally-occurring TIP,
as well as
synthetic or recombinant TIP (rec TIP). Further, the term "tubular
infundibular peptide"
encompasses allelic variants, species variants, and conservative amino acid
substitution variants.
The term also encompasses full-length TIP-(1-39), as well as TIP fragments. It
will thus be
understood that fragments of TIP variants, in amounts giving equivalent
biological activity to
TIP-(1-39), can be-used in the methods of the invention, if desired. Fragments
of TIP
incorporate at least the amino acid residues of TIP necessary for a biological
activity similar to
that of intact TIP-(1-39). Examples of such fragments are TIP-(1-29), TIP-(1-
30), TIP-(1-31), ...
TIP-(1-37), TIP-(1-38), and TIP-(1-39).
The term "tubular infundibular peptide" also encompasses variants and
functional
analogues of TIP having an homologous amino acid sequence with TIP-(1-39). The
present
invention thus includes pharmaceutical formulations comprising such TIP
variants and functional
analogs, carrying modifications like substitutions, deletions, insertions,
inversions or
cyclisations, but nevertheless having substantially the biological activities
of TIP-(1-39).
The calculation of % homology and % identity are determined by first aligning
a
candidate TIP polypeptide with SEQ ID NO:26, as provided in FIG. 3.
"Homologous amino
acid sequence" means an amino acid sequence that differs from an amino acid
sequence shown
in SEQ ID NO: 15, by one or more conservative amino acid substitutions, or by
one or more
non-conservative amino acid substitutions, deletions, or additions located at
positions at which
they do not destroy the biological activities of the polypeptide. Preferably,
such a sequence is at
least 75%, preferably 80%, more preferably 85%, more preferably 90%, and most
preferably
95% homologous to the amino acid sequence in SEQ ID NO: 26. Homologous amino
acid
sequences also include sequences that are identical or substantially identical
to an amino acid
sequence as shown in SEQ ID NO: 26. By "amino acid sequence substantially
identical" is
meant a sequence that is at least 60%, preferably 70%, more preferably 80%,
more preferably
90%, and most preferably 95% identical to an amino acid sequence of reference.
Preferably the
homologous sequence differs from the reference sequence, if at all, by a
majority of conservative
amino acid substitutions.
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The methods of the present invention includes the use of a TIP peptide
selected from the
group consisting of:
(a) full-length TIP;
(b) biologically-active variants of full-length TIP;
(c) biologically active TIP fragments;
(d) biologically active variants of TIP fragments;
(e) biologically active variants having at least 75% homology with SEQ ID
NO:26;
(f) biologically active variants having at least 60% identity with SEQ ID
NO:26; and
(g) biologically active variants encoded by a nucleic acid sequence that
hybridizes
under stringent conditions to a complementary nucleic acid sequence of SEQ ID-
NO:25.
D. FORMULATIONS AND METHODS OF TREATMENT
Compositions of the present invention (i.e., PTHrP peptide, and the skeletal
anabolic
agents described above) may be administered intermittently by any route which
is compatible
with the particular molecules and, when included, with the particular bone
resorption inhibiting
agent. Thus, as appropriate, administration may be oral or parenteral,
including subcutaneous,
intravenous, inhalation, nasal, and intraperitoneal routes of administration.
In addition,
intermittent administration may be by periodic injections of a bolus of the
composition once
daily, once every two days, once every three days, once weekly, twice weekly,
biweekly, twice
monthly, and monthly
Therapeutic compositions of the present invention may be provided to an
individual by
any suitable means, directly (e.g., locally, as by injection, implantation or
topical administration
to a tissue locus) or systemically (e.g., parenterally or orally). Where the
composition is to be
provided parenterally, such as by intravenous, subcutaneous, intramolecular,
ophthalmic,
intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital,
intracerebral, intracranial,
intraspinal, intraventricular, intrathecal, intracisternal, intracapsular,
intranasal or by aerosol
administration, the composition preferably comprises part of an aqueous or
physiologically
compatible fluid suspension or solution. Thus, the carrier or vehicle is
physiologically
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acceptable so that in addition to"de1'iver'y""o"f the desired composition to
the patient, it does not
otherwise adversely affect the patient's electrolyte and/or volume balance.
The fluid medium for
the agent thus can comprise normal physiologic saline (e.g., 0.9% aqueous
NaCl, 0.15 M, pH
7-7.4). Alternatively, the use of pulsatile administration of the skeletal
anabolic drug by mini-
pump can be employed in the methods of the present invention.
Useful solutions for parenteral administration may be prepared by any of the
methods
well known in the pharmaceutical art, described, for example, in REMINGTON' S
PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), Mack Pub., 1990. Formulations of
the
therapeutic agents of the invention may include, for example, polyalkylene
glycols such as
polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like.
Formulations for direct administration, in particular, may include glycerol
and other
compositions of high viscosity to help maintain the agent at the desired
locus. Biocompatible,
preferably bioresorbable, polymers, including, for example, hyaluronic acid,
collagen, tricalcium
phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide
copolymers, may
be useful excipients to control the release of the agent in vivo. Other
potentially useful parenteral
delivery systems for these agents include ethylene-vinyl acetate copolymer
particles, osmotic
pumps, implantable infusion systems, and liposomes. Formulations for
inhalation administration
contain as excipients, for example, lactose, or may be aqueous solutions
containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily
solutions for
administration in the form of nasal drops, or as a gel to be applied
intranasally. Formulations for
parenteral administration may also include glycocholate for buccal
administration,
methoxysalicylate for rectal administration, or cutric acid for vaginal
administration.
Suppositories for rectal administration may also be prepared by mixing the
PTHrP peptide (alone
or in combination with a bone resorption-inhibiting agent) with a non-
irritating excipient such as
cocoa. butter or other compositions that are solid at room temperature and
liquid at body
temperatures.
Formulations for topical administration to the skin surface may be prepared by
dispersing
the molecule capable of releasing the PTHrP peptide (alone or in combination
with a bone
resorption-inhibiting agent, or an anabolic agent) with a dermatologically
acceptable carrier such
as a lotion, cream, ointment or soap. Particularly useful are carriers capable
of forming a film or
layer over the skin to localize application and inhibit removal. For topical,
administration to
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internal tissue surfaces, the agent may be dispersed in a liquid, or semisolid
tissue adhesive or
other substance known to enhance adsorption to a tissue surface, e.g., a bone
paste. For example,
hydroxypropylcellulose or fibrinogen/thrombin solutions may be used to
advantage.
Alternatively, tissue-coating solutions, such as pectin-containing
formulations may be used.
The method of treatment can constitute a single period of intermittent
administration of a
skeletal anabolic drug (e.g., for a period of time varying between 1-3 months
to 15-18 months).
The period of administration is preferably 12, 15, or 18 months, more
preferably 7, 8, 9, 10, or
11 months, and most preferably 1, 2, 3, 4, 5, or 6 months. Alternatively, in
another embodiment,
the method of treatment can constitute a series of administration periods
followed by periods of
no administration (e.g., sequential periods of three months of intermittent
administration of a
skeletal anabolic drug and three months of no drug administration). The
sequential treatment
periods can be repeated until the patient BMD is restored (e.g., a T-score <-
2.0 or -2.5 below
the mean or preferably <-1.0 below the mean).
In yet another embodiment, the method of treatment further includes the step
of
co-administering, either simultaneously or sequentially to said patient a bone
resorption
inhibiting ageint. The bone resorption-inhibiting agent can be a
bisphosphonate, estrogen, a
selective estrogen receptor modulator, a selective androgen receptor
modulator, calcitonin, a
vitamin D analog, or a calcium salt. The bone resorption-inhibiting agent can
also be
alendronate, risedronate, etidronate, pamidronate, tiludronate, zoledronic
acid, raloxifene,
tamoxifene, droloxifene, toremifene, idoxifene, levormeloxifene, or conjugated
estrogens. In
one embodiment, the patient receives intermittent administration of the
skeletal anabolic drug for
a period of time, followed by a period of treatment with a bone resorption
inhibiting agent, either
alone or in combination with the skeletal anabolic drug. In a currently
preferred embodiment, an
anabolic agent such as PTHrP is first administered, for example, over a three
month period or
longer, followed by administration of an antiresorptive agent either alone or
in combination with
the skeletal anabolic drug, for example, over an additional three month period
or longer.
Without being restricted to theory, reverse administration, i.e., giving the
antiresorptive agent
before administration of the anabolic agent, diminishes the efficacy of the
anabolic agent.
Hence, according to the present invention, anabolic agents such as PTHrP
should be the primary
osteoporosis therapeutics, with antiresorptives used later to maintain and
enhance the
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PTHrP/PTH/TIP effects, and for example, estrogen or bisphosphonates
osteoporosis
administered as second line agents after the anabolics.
However, a skilled artisan will recognized that the sequential treatment
regimen could
begin with a treatment period with a bone resorption inhibiting agent followed
by a treatment
period with the skeletal anabolic drug, that the leiigth of sequential
treatment periods can be
modified (e.g., 1-18 months), and that the skeletal anabolic drug can be co-
administered with the
bone resorption inhibiting agent (e.g., sequential treatment period of a
skeletal anabolic drug and
a bone resorption inhibiting agent followed by a treatment period of a bone
resorption inhibiting
agent alone). Again, as stated above, the sequential treatment periods (e.g.,
three months of the
skeletal anabolic drug followed by three mointh of the bone resorption
inhibiting agent) can be
repeated until the patient BIVID is restored (e.g., a T-score <-2.0 or -2.5
below the mean or
preferably <-1.0 below the mean).
Skeletal anabolic agents are commonly believed to demonstrate numerous adverse
side
effects, and as a result, the dosage and administration of these agents is
carefully controlled, and
the patient carefully monitored for emergence of unwanted side effects. For
example, PTHrP
was originally thought to be responsible for most instances of hypercalcemia
of malignancy, a
syndrome that resembles hyperparathyroidism, with a toxicity profile believed
to be similar to or
even greater to that of PTH.
However, the toxicity profiles of other skeletal anabolic agents do not appear
to be
applicable to PTHrP. The fmdings of the present invention indicate that
despite being
administered in doses, for example, at least 20 times higher than those
considered safe for PTH,
PTHrP does not cause significant side effects. For example, intermittent doses
of PTHrP of
about 50 micrograms to about 400 micrograms given subcutaneously (Q2H for 8
hours after a
dose), does not appear to cause hypercalcemia. In fact, administration of
PTHrP has never been
observed to cause hypercalcemia at any dose yet given, such as doses exceeding
450 micrograms
and up to 1 milligram are safe, well tolerated by patients and efficacious. In
certain cases,
individual doses of 3-10 milligrams appear safe, and even up to 50 mg or
greater appear well
tolerated and are also possible given proper patient monitoring.
Specifically, there have been no examples of the development of hypercalcemia
(defined
in the studies described in Example 1 and Example 5 as a serum calcium above
9.9 mg/dl, a very
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conservative defmition of hypercalcemia) in 18 PTHrP-treated patients despite
the comparatively
higher doses employed. This contrasts with the Neer, et al. study
demonstrating an 11 %
incidence of hypercalcemia reported among patients treated with PTH at the 20
microgram dose
and a 28% incidence of hypercalcemia reported among patients who received the
40 microgram
dose. Interestingly, Neer, et al. defined hypercalcemia as a serum calcium
greater than 10.6
mg/dl. Recalculation of the results of the Neer, et al study using the more
rigorous 9.9 mg/dl
criteria for hypercalcemia described herein, would have resulted in a much
higher hypercalcemia
incidence in the Neer, et al. study. Other researchers have seen even more
severe hypercalcemia,
up to 15 mg/dl, which is near lethal, using PTH(1-84) at doses of
approximately 40 micrograms.
Thus, PTHrP offers many advantages over PTH as a therapeutic. It is a pure
anabolic
skeletal agent which is non-hypercalcemic, and has no other adverse effects
even when
administered in the comparatively higher doses explored to date. Second it
appears far more
efficacious than PTH in increasing bone mass density. - Third, it is more
stable than PTH.
Fourth, it has markedly different and more favorable pharmacokinetics than
PTH. Fifth, it is
responsible for maintaining bone mass in adults, in contrast to PTH, which is
not required to
maintain bone mass. Sixth, it can achieve therapeutic endpoints in shorter
time-frames, and is
thereby safer for human administration, for example use for only 3-9 months
can achieve
dramatic,effects on BMD without crossing the 12-month osteosarcoma threshold.
E. BIOASSAY OF ANABOLIC EFFICACY OF PTHRP ANALOGS
The synthesis, selection and use of PTHrP or analogs thereof and other
anabolic agents,
which are capable of promoting borie formation, are within the ability of a
person of ordinary
skill in the art. For example, well-known in vitro or in vivo assays can be
used to determine the
efficacy of various candidate PTHrP analogs to promote bone formation in human
patients. For
in vitro binding assays, osteoblast-like cells which are permanent cell lines
with osteoblastic
characteristics and possess receptors for PTHrP of either rat or human origin
can be used.
Suitable osteoblast-like cells include ROS 17/2 (Jouishomme et al.,
Endocrinolog.y, 130: 53-60
(1992)), UMR 106 (Fujimori et al., Endocrinology, 130: 29-60 (1992)), and the
human derived
SaOS-2 (Fukuyama et al., Endocrinology, 131: 1757-1769 (1992)). The cell lines
are available
from American Type Culture Collection, Rockville, Md., and can be maintained
in standard
specified growth media. Additionally, transfected human embryonic kidney cells
(HEK 293)
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expressing the human PTH1 or PTH2 receptors can also be utilized for in vitro
binding assays.
See, Pines et al., Endocrinology, 135: 1713-1716 (1994).
For in vitro functional assays, PTHrP -like analog activities of peptide
fragments or
derivatives of PTHrP can be tested by contacting a concentration range of the
test compound
with the cells in culture and assessing the stimulation of the activation of
second messenger
molecules coupled to the receptors, e.g., the stimulation of cyclic AMP
accumulation in the cell
or an increase in enzymatic activity of protein kinase C, both of which are
readily monitored by
conventional assays. See, Jouishomme et al., Endocrinology, 130: 53-60 (1992);
Abou-Samra et
al., Endocrinology, 125: 2594-2599 (1989); Fujimori et al., Endocrinology,
128: 3032-3039
(1991); Fukayama et al., Endocrinology, 134: 1851-1858 (1994); Abou-Samra et
al.,
Endocrinology, 129: 2547-2554 (1991); and Pines et al., Endocrinology, 135:
1713-1716 (1994).
Other parameters of PTH action include increase in cytosolic calcium and
phosphoinositols, and
biosynthesis of collagen, osteocalcin, and alteration in alkaline phosphatase
activity.
Agonist activities of subfragments of PTH have been successfully analyzed by
contacting
peptides with rat kidney cells in culture and assessing cyclic AMP
accumulation (Blind et al.,
Clin. Endocrinol., 101: 150-155 (1993)) and the stimulation of 1,25-
dehydroxyvitamin D3
production (Janulis et al., Endocrinology, 133: 713-719 (1993)).
As demonstrated in Examples 2 and 3 below, PTH and PTHrP with bone formation
activity bind specifically with PTH/PTHrP receptors and produce a dose-
dependent stimulation
of cAMP accumulation in human renal cortical membranes, in human osteoblast-
like
osteosarcoma membranes and intact cells (Example 2), and in canine renal
cortical membranes
(Example 3). With [Nle8 18,Tyr34] hPTH-(1-34) NH2 or hPTHrP-(1-36) as the
reference standard
analogs, a dose-response relationship can be generated using standard non-
linear regression
analysis. The relative potency for various PTHtP analogs (in units/mg) can be
determined from
the ratio of the EC50 of the reference standard analog to that of the PTHrP
analog. EC50 is
defined as the dose that evokes a half-maximal response-cAMP accumulation
herein. The
detailed procedure for handling the cells; setting up the assay, as well as
methods for cAMP
quantitation, is described in Sistane et al., Pharmacopeial Forum 20: 7509-
7520 (1994).
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For in vivo assays, candidate PTHrP analogs can be characterized by their
abilities to
increase trabecular and cortical bone mass in ovariectomized, osteopenic rats,
as described in
Example 4.
Example 5 describes a three-month double blind, prospective, placebo-
controlled
randomized clinical trial, demonstrating the effectiveness of PTHrP as a
skeletal anabolic agent.
PTHrP displays minimal side effects, for example, despite the comparatively
high doses, no
significant increase in hypercalcemia is observed.
Example 6 describes a computer system and methods of using the same, for
structural
based design of peptidomimetics and small molecules having skeletal anabolic
biological
activity.
The following examples are provided for illustrative purposes only, and are in
no way
intended to limit the scope of the present invention.
EXAMPLE 1 SHORT-TERM, VERY HIGH-DOSE TREATMENT OF
POSTMENOPAUSAL OSTEOPOROSIS WITH THE SKELETAL
ANABOLIC AGENT PTHRP
Parathyroid hormone-related protein,-or "PTHrP", is the quintessential
skeletal catabolic
agent. It was initially discovered as the cause, of the common lethal
paraneoplastic syndrome,
humoral hypercalcemia of malignancy or "HHM". Hypercalcemia occurring among
patients
with HHM results principally from a striking activation of osteoclastic bone
resorption. Thus,
PTHrP would seem an unlikely candidate as a skeletal anabolic agent.
The purpose of the present study was to determine whether the administration
of
intermittent high doses of a PTHrP peptide, for a short period of time could
produce significant
increases in BMD without negative side effects, and that as such, PTHrP might
be an effective
skeletal anabolic agent in women with postmenopausal osteoporosis. Reasoning
that parathyroid
hormone (PTH) can cause demonstrable increases in bone mineral density within
three months
of treatment, and that PTHrP would need to be at least as effective as PTH in
increasing bone
mass to be useful therapeutically, the study described herein as Example 1 is
three month double-
blinded, randomized placebo-controlled pilot clinical trial in which PTHrP was
compared to
placebo treatment.
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The rate of increase, as well as the absolute increase, observed in lumbar
spine bone
mineral density with PTHrP are large, and may equal or exceed those reported
to date using
currently available osteoporosis drugs.
PTHrP administered subcutaneously in high doses for only three months appears
to be a
potent anabolic agent, producing a 4.7% increase in lumbar spine BMD. This
compares very
favorably to available anti-resorptive drugs for osteoporosis, and PTH.
Despite the high doses,
PTHrP was well tolerated.
Materials and Methods
Preparation of hPTHrP-(1-36) and placebo for human injection
Synthetic hPTHrP-(1-36) was prepared by solid-phase synthesis, as previously
described
(Everhart-Caye et al. J Clin Endocrinol Metab 81: 199-208 (1996)).
Briefly, hPTHrP-(l-36) was weighed, dissolved in 10 mM acetic acid, filtered
through a
sterile 0.2 m syringe filter, aseptically aliquoted into 5-600 g aliquots in
sterile glass vials,
aseptically sealed into the vials, frozen at-80 C, and lyophilized. Placebo
vials were prepared
in exactly the same manner. The vials were stored -at -80 C. Peptide content
was confirmed by
amino acid analysis, PTHrP-(1-36) RIA (described below) or PTHrP-(1-36) RNA,
and adenylyl
cyclase bioassay (described below). Pyrogen testing was performed by limulus
amebocyte lysate
gel-clot assay method (Associates of Cape Cod, Falmouth, MA), using standard
endotoxin from
Escherichia coli 0113 as a control. The endotoxin concentration in the vials
was below the
lower limit of detection (<0.03 endotoxin units/mL). The vials were labeled in
coordination with
the University of Pittsburgh Medical Center Investigational Pharmacy.
Immediately previous to
the beginning of each injection, the PTHrP-(1-36) from a vial was
reconstituted in 1.0 mL 0.9%
saline. The mass of hPTHrP (1-36) is 4260.6 Da. The structures of the peptides
were confirmed
by mass spectroscopy and amino acid analysis. Greater than 99% purity was
confirmed by
reverse-phase high performaince liquid chromatography.
Adenylyl cyclase bioassay
The biological potency of hPTHrP-(1-36) was tested using an adenylyl cyclase
assay
performed in confluent SaOS2 human osteosarcoma cells, using a method
previously described.,
in detail (Everhart-Caye et al. J Clin Endocrinol Metab 81: 199-208 (1996);
Orloff et al.
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Endocrinol 131: 1603-1611 (1992); Merendino et al. Science 231: 388-390
(1986)). Briefly,
SaOS2 cells were obtained from American Type Culture Collection, Rockville,
MD, and were
maintained in McCoy's medium supplemented with 10% FBS, 2 mmol/L L-glutamine,
penicillin
(50 U/mL), and streptomycin (50 g/mL). Cells were plated approximately 10
days before assay
in 24-well plates and had been confluent for approximately 7 days previous to
assay. The cells
were incubated at 25 C with isobutylmethylxanthine (500 mmol/L) for 10 min,
the peptides
added, and the incubation continued at 25 C for another 10 min. Medium was
aspirated, the
cells solubilized in 5% trichloroacetic acid, and the extracts neutralized
using 25:75%
trioctylamine:Freon. Content of cAMP in the extracts was measured by RIA
(Biomedical
Technologies, Stoughton, MA). The peptide was examined in at least three
different assays.
PTHrP RIA
The hPTHrP-(1-36) RIA using antiserum S2 has been described in detail
previously
(Yang et al., Biochem., 33: 7460-7469 (1994); Burtis et al.,1V. Engl. J. Med.,
322: 1106-1112
(1990)). Briefly, the lactoperoxidase method was used to prepare 1asI-labeled
Tyr36PTHrP-
(1-36) amide for use as radioligand (see below) as described previously
(Orloff et al. J. Biol.
Chem., 264: 6097-6103 (1989)). Duplicates of assay standard or sample (100 L)
were
incubated overnight at 4 C with (100 L) of a 1:1500 dilution of S-2 in P10BT
buffer (PBS
containing 10% BSA and 0.1% Triton X-100). Iodinated Tyr36 of a 1:1500
dilution of S2 in
P10BT buffer (PBS containing 10% BSA and 0.1% Triton X-100). Iodinated
Tyr36hPTHrP-
(1-36)amide (2000-8000 cpin) in PBT buffer was added to the tubes, and the
mixture was
incubated overnight at 4 C. Phase separation was accomplished using dextran-
coated charcoal.
The sensitivity of the assay is 50 pmol/L. The antiserum recognizes hPTHrP-(1-
74), (1-36) and
(1-141) with equal affinity, but fails to cross-react with hPTHrP-(37-74) or
with hPTH-(1-34) or
hPTH-(1-84) (Yang et al., Biochem., 33: 7460-7469 (1994)).
Serum and Urine Biochemistries
Blood was analyzed for routine chemistry and hematology studies in the
University of
Pittsburgh Medical Center Clinical Chemistry Laboratory, as were plasma 25-
vitamin D
concentrations. Osteocalcin was measured as described in Gundberg, et al., J
Clin Endocrinol
Metab 83:3258-3266, (1998), incorporated by reference. Serum N-telopeptide (N-
Tx)
(Osteomark) and urinary deoxypyridinolines (DPD) (Pyrilinks-D) were measured
using
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commercial kits from Ostex International, Seattle WA, and Quidel Corp, Santa
Clara CA,
respectively.
Study subjects
Sixteen consecutive healthy postmenopausal women with osteoporosis were
identified
for this study. All study subjects provided informed consent. The participants
in the
experimental and control groups were of similar age (mean age approximately
65), weight,
height, BMI, years since menopause, years on estrogen, calcium intake, and had
similar plasma
25 vitamin D concentrations. Both groups displayed osteoporosis at the lumbar
spine.
Before beginning the study, each subject underwent a bone mineral density scan
(DXA)
of the lumbar spine and hip at the beginning and at the conclusion of the
study. Inclusion criteria
included a T-score of less than -2.5 at the lumbar spine; being more than
three years
postmenopausal, being on estrogen replacement for at least three years, and
being in generally
excellent health. Exclusion criteria included use in the past of any
osteoporosis medication,
including bisphosphonates, calcitonin, or selective estrogen receptor
modifiers. Current use of
medications or agents that might influence calcium or bone metabolism (e.g.,
thiazides, non-
physiologic doses of thyroid hormone, glucocorticoids, lithium, alcohol, etc.)
was also an
exclusion criterion. All study subjects provided informed consent. The
protocol was approved
by the University of Pittsburgh Institutional Review Board.
Study protocol
The use of PTHrP in human clinical trials was approved by the FDA (IND #
49175,
incorporated herein by reference). The protocol was approved by the University
of Pittsburgh
Institutional Review Board. This was a randomized, double-blinded placebo-
controlled clinical
trial. The primary outcome measure was lumbar spine bone mineral density.
Secondary
outcome measures were hip and femoral neck bone mineral density, markers of
bone turnover,
serum calcium, serum creatinine, renal phosphorus handling and adverse events.
The sixteen subjects were randomized to receive three months of treatment with
either
PTHrP or placebo (identically prepared empty vials containing no PTHrP). Each
subject also
received 400 IU of vitamin D and 1000 mg of calcium as calcium carbonate per
day (Os-Cal,
Smith Kline Beecham/Glaxo, King of Prussia, PA), and this was started two
weeks before the
initiation of PTHrP or placebo treatments. Subjects were taught in the home
storage at -20 C,
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reconstitution and self-injection of PTHrP or placebo. Vials were
reconstituted by the study
subjects in 1.0 ml of sterile bacteriostatic saline immediately prior to use,
to an average dosage of
PTHrP of 410.25 g per day, or saline placebo, and was self-administered into
the abdominal
subcutaneous fat. Subjects returned for blood and urine studies at 0, 14, 30,
60 and 90 days of
the study. A fmal bone density study was performed on day 90 of the study.
Study Compliance
One patient in the placebo group dropped out of the study after three days.
The
remaining subjects in each group completed the study without event. The data
analysis which
follows includes all 16 patients at baseline, and the eight PTHrP and seven
placebo subjects who
completed the three months of the study.
Safety considerations
Study subjects were monitored at 0, 2, 4, 8, and 12 weeks for hypercalcemia,
rashes, GI
complaints, cardiovascular complaints or symptoms, or other non-specific
complaints. Subjects
were questioned regarding adverse effects at each visit, i. e., 0, 14, 30, 60
and 90 days of the
study.
Bone Densitometry
Bone densitometry at the spine and hip was measured blindly using a Model 2000
densitometer (Hologic Inc. Bedford MA). The results were blindly and
independently reviewed
by two physicians experienced bone densitometrists.
Statistical analysis
Statistical analysis was performed using Student's unpaired T-test, using
Excel software
(Microsoft, Seattle, WA). P-values less than 0.05 are considered significant.
Results
Baseline Demographics.
The baseline demographics in the two groups are shown in Table I. The subjects
were of
similar age, weight, height, BMI, years since menopause, years on estrogen,
calcium intake, and
had similar plasma 25 vitamin D concentrations. In the placebo group, two were
smokers and
one was on a normal replacement dose of thyroid hormone for hypothyroidism.
Both groups
displayed osteoporosis at the lumbar spine.
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Study Compliance.
One patient in the placebo group dropped out of the study after three days
because of
shortness of breath and chest tightness following a subcutaneous injection.
The remaining
subjects in each group completed the study without event. The following data
analysis includes
all 16 patients at baseline, and the eight PTHrP and seven placebo subjects
who completed the
three months of the study.
Primary Outcome
L/S BMD
The changes in BMD at the lumbar spine over the three months of the study are
shown in
FIG. 4. The left panel shows the changes in bone mineral density as measured
by DXA as
percent changes from baseline. The right panel shows the same data as absolute
changes in bone
mineral density from baseline in gm/cm2. In each panel, the bold line
represents the subjects
treated with PTHrP (n = 8 indicates that all eight PTHrP treated patients are
included), and the
dotted line, those receiving placebo.
In the placebo group, the data are presented including the outlier (+) and
with the outlier
excluded (-), as described in the text (n = 6/7 indicates the numbers of
subjects receiving
placebo including or excluding he outlier). The error bars represent SEM. P-
values were
determined using Student's paired T-test. As can be seen in the left panel,
the increase in BMD
at the lumbar spine in the PTHrP group was 4.72% over three months. In
contrast, the change in
the placebo group was smaller, 1.4%, p = 0.025. This surprisingly large
increment in the placebo
group reflected a 6.5% increase in one subject. The reason for the marked
increase, 6.5%, in
BMD in the single placebo outlier is unknown. The increase was confirmed by
independent
blinded review of DXA scans, and was not due to positioning or other technical
considerations.
This subject was no different than the other placebo subjects in total hip or
femoral neck BMD at
baseline or at the conclusion, and was no different with regard to baseline
spine BMD. There
was no evidence of a vertebral compression fracture before or after the study,
and there was no
aortic or arthritic calcification. This subject had one of the lowest plasma
25 vitamin D
concentrations in the study (16 ng/ml), and it is possible that a component of
this subject's
marked increase reflected correction of mild osteomalacia. If this subject is
excluded, the
increase in the placebo group was 0.6%, p= 0.003. Similar fmdings were
obtained when the
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results are expressed as absolute changes in BMD in grams per cm2 (right
panel), with the
increment in the PTHrP group being 0.0375 gm/cm2, and 0.011 or 0.005 gm/cm2 in
the placebo
group, depending on whether the outlier is included (p = 0.022) or excluded
(0.003).
Secondary Outcomes
Femoral Neck and Total Hip BMD
The changes in BMD expressed as percent change from baseline at the total hip
and
femoral neck are shown in FIG. 5, and are compared to the changes at the
lumbar spine. The
light gray bars indicate the placebo group (PBO), and the black bars indicate
the experimental
group (PTHrP). The L/S data are the same as those presented in FIG. 4 and
include the outlier.
The error bars indicate SEM, and P-values were determined using Student's
paired T-test. There
was no significant difference between the PTHrP or PBO groups at either hip
site during the
study.
Bone turnover markers
FIG. 6 illustrates three different bone turnover markers in the placebo and
PTHrP-treated
subjects. FIG. 6(a) illustrates serum osteocalcin, a marker of bone formation,
increased in a
statistically significant fashion during the study in the PTHrP-treated
subjects but not the placebo
controls. Indeed, as illustrated in FIG. 6(a), increases in serum osteocalcin
were apparent as
early as day 15 (the earliest time period blood samples were obtained).
In contrast, serum NTX, a marker of bone resorption, remained unchanged during
the
study in the PTHrP-treated subjects, as it did in the placebo controls, as
shown in FIG. 6(b).
Urinary DPD excretion, a second marker of bone resorption, was also unchanged,
see, FIG. 6(c).
In all three figures, the dotted line indicates the placebo group and the sold
line the PTHrP group.
The error bars indicate SEM, and the P-values were determined using ANOVA for
repeated
measures. These findings suggest that PTHrP selectively stimulates bone
formation without
further stimulating normal rates of bone resorption.
Serum and urine chemistr=ies
FIG. 7 illustrates serum total and ionized serum calcium in the placebo and
PTHrP-
treated subjects. The dotted line indicates the placebo group and the sold
line, the PTHrP group.
The error bars indicate SEM, and the P-values were determined using ANOVA for
repeated
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measures. Calcium levels remained normal and constant in both the PTHrP-
treated subjects as
well as in the placebo controls. No subject developed a significant increase
in serum total or
ionized calcium. Serum creatinine remained normal as well in both the PTHrP
and placebo
subjects (mean serum creatinine, SEM, on day 90 = 0.825 0.05 mg/dl in the
PTHrP group vs.
0.84 0.06 in the placebo group, p = ns). Serzun phosphorus was also similar
in both groups
throughout the study (3.2 mg/dl 0.18 in the PTHrP group vs. 2.9 0.17 in
the placebo group,
p = ns), as was the tubular maximum for phosphorus (3.3 mg/dl 0.27 in the
PTHrP group vs.
2.6 + 0.24 in the placebo group, p = ns).
FIG. 8 illustrates a comparison of the anabolic activity of PTHrP with results
from
selected previously published osteoporosis clinical trials. "Ralox 150" refers
to Delmas PD, et
al., N Engl J Med 337:1641-7, (1997); "Ralox 120" to Ettinger B, et al., JAMA
282:637-45,
(1999); and "calcitonin" to Chestnut C, et al., Osteoporosis Int 8(suppl 3):13
(1998); "alendro",
"risedro" and "zoledro" refer to studies employing alendronate (Liberman UA,
et al., N Engl J
Med 333:1437-43, (1995) and Murphy MG, et al., J Clin Endocrinol Metab 86:1116-
25, (2001),
to risedronate (Fogelman I, et al., J Clin Endocrinol Metab., 85:1895-1900,
(2000) and McClung
MR, et al, N Engl J Med 344:333-40, (2001), and to zoledronate, Reid IR, et
al., N Engl J Med
346:653-61, 2002.). "PTH" refers to two studies employing parathyroid hormone,
Lindsay R, et
al., Lancet 350:550-5 (1997), and Neer RM, et al., N Engl J Med 344:1434-41
(2001), and
"PTHrP" refers to the current study. Each of the foregoing references are
hereby incorporated
herein by reference in their entirety.
Aa'verse events
No subject in the PTHrP group experienced weakness, nausea, vomiting,
diarrhea,
constipation, flushing, muscle cramps or allergic phenomena. One PTHrP subject
experienced
seconds of heart palpitations with standing after the third injection, which
did not recur with
25 subsequent injections. All PTHrP subjects completed the study. In contrast,
one subject in the
placebo group experienced flushing, dizziness and nausea after her injection
on day three of the
study, and this subject withdrew from the study.
Discussion
These studies indicate that PTHrP, administered subcutaneously in very large
doses over
30 a very brief period of time, can cause statistically and biologically
important increments in spine
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bone density. This is surprising tor a number of reasons. First, PTHrP was
originally identified
as a result of its skeletal catabolic actions in humoral hypercalcemia of
malignancy. Second, the
rate and absolute increment in spine BMD, almost 5% in three months, is larger
than those
observed using many currently available anti-resorptive osteoporosis
medications, (see, FIG. 8).
Indeed, increments of this magnitude have never been reported using calcitonin
nor raloxifene,
even when these agents are given for as long as three years. Estrogen causes
similar increments
in spine BMD, but a change of 5% requires three years of treatment. The
changes observed
using some bisphosphonates, including etidronate, alendronate, risedronate,
and zoledronate,
may equal or exceed 5%, but require far longer than three months, typically
one or more years.
Indeed, the changes observed compare favorably to, and may possibly exceed
those observed in
studies reported to date using PTH over a three month period. Viewed from the
perspective of
available anti-resorptive therapies, the effects of short-term high dose PTHrP
are striking.
The doses of PTHrP employed in this study were large compared to those used in
similar
PTH studies. Subjects in this study received 6.56 micrograms/kg/day, which on
average was
410.25 micrograms per day in the eight subjects who received PTHrP. This is
some 10- to 20-
fold larger than doses of hPTH(1-34) (20-40 micrograms/day) commonly employed
in
osteoporosis studies. Doses of PTH in excess of 20 micrograms/day are
associated with
hypercalcemia and other adverse effects in humans. It is surprising,
therefore, that healthy
subjects would tolerate doses of this magnitude without developing
hypercalcemia, postural
hypotension, nausea, flushing or other adverse effects. The differences cannot
be ascribed to
differences in molar amounts of the two peptides employed, for PTHrP(1-36) is
very close in
molecular weight to PTH(1-34) (approximately 4200 Mr). Nor can the differences
be ascribed to
different interactions with the common PTH/PTHrP receptor: both hPTH(1-34) and
hPTHrP(1-
36) display similar or identical binding kinetics and signal transduction
activation characteristics.
Importantly, in head-to-head comparison with hPTH(1-34) in vitro and also in
vivo given
intravenously to human volunteers, PTHrP(1-36) is equal in potency to hPTH(1-
34). Different
serum metabolic clearance rates are an unlikely explanation as well, for we
have demonstrated
that the T1i2 of intravenously infused PTHrP(1-36) is six minutes,
indistinguishable from the five
to six minutes reported for hPTH(1-34).
The differences in skeletal effects of the two peptides relate to differing
pharmacokinetic
characteristics of PTH and PTHrP following subcutaneous injection. Human PTH(1-
34) has
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been reported in two studies to reach peak plasma levels at 30-45 minutes
following injection,
whereas we have reported that peak plasma levels of PTHrP occur at or before
15 minutes
following a subcutaneous dose. Indeed, since the 15 minute time point was the
first we
examined, and since circulating PTHrP values appeared to be in a sharp decline
at this initial 15
minute time point, it is very likely that the peak occurs much earlier,
perhaps at five to ten
minutes. Thus, hPTHrP(1-36) is absorbed more rapidly than PTH following
subcutaneous
injection, and plasma levels of PTHrP reach their peak and therefore decline
more rapidly than
those of PTH.
The different absorption and clearance kinetics of PTHrP vs. PTH underlie the
requirement for large dose of PTHrP as well as the lack of hypercalcemia and
other toxicities
observed in the patients studied despite these large doses. This apparent
safety is supported by
our a prior studies in which an additional seven subjects received the same
6.56
micrograms/kg/day dose for two weeks with no adverse events, and another study
in which this
dose was administered as a single dose to three health individuals. Thus, no
adverse events have
been encountered in a total of 18 healthy human subjects receiving these large
doses of PTHrP
for periods of one day, two weeks or three months.
Mechanistically, the bone turnover marker data (see, FIG. 6(a), (b), and (c))
suggest that
PTHrP may have purely anabolic effects on the skeleton, without the
accompanying increase in
bone resorption observed using PTH. Thus, in contrast to PTH, which displays
both formation-
and resorption-stimulating properties, PTHrP appears to have selective
osteoblastic or anabolic
effects, without concomitant resorption-stimulating effects. The lack of a
resorptive effect is
unlikely to be due to concomitant estrogen use since the resorptive response
to PTH is not
abolished by estrogen. Interestingly, while the rate of increase in BMD in the
current study was
very large, the total increase in the formation marker osteocalcin was either
similar to, or
significantly lower than that reported using PTH. The apparent relatively
lower increase in
formation, in the setting of a rather dramatic increase in BMD, supports the
biochemical
evidence for an apparent lack of a resorption-stimulating effect. Confirmation
of these finding
can be made using skeletal biopsies and quantitative bone histomorphometry.
The lack of a resorptive effect is not likely due to the brief duration (three
months) of
administration of PTHrP, since prior studies have shown that PTH increases
bone resorption
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significantly at or well before three months. For example, in a study Lindsay
et al. (Lancet 350:
550-555 (1997)), resorption as assessed using urinary NTX, was already
elevated at two weeks,
and was increased by 25% at three months. Finkelstein et al. (NEngl JMed 331:
1618-1623
(1994)) demonstrated that urinary hydroxyproline and pyridinolines, two
different markers of
bone resorption, were increased by approximately 200% at three months
following treatment
with PTH. Similarly, Hodsman (J Clin Endocrinol Metab 82: 620-628 (1997)) has
demonstrated
that both urinary hydroxyproline and NTX are significantly increased by only
four weeks of
treatment using PTH.
Similarly, the lack of a resorptive effect is unlikely to be due to
concomitant estrogen use.
First, the same type of dissociation was observed in our earlier study in
postmenopausal women
without estrogen use (Plotkin et al., J Clin Endocrinol Metab 83: 2786-2791
(1998)). Second, the
resorptive.response to PTH is easily apparent in estrogenized women in both
the Roe and the
Lindsay studies at three months (Roe et al., Program and Abstracts of the 81
st Annual Meeting
of the Endocrine Society, San Diego, CA, June 12-15, 1999, p. 59; Lindsay et
al., Lancet 350:
550-555 (1997)). Thus, from the data available to date, it appears that PTHrP,
in the doses
employed thus far, and for the duration observed to date, may be different
from PTH and may
display purely anabolic affects.
Assuming that the selective anabolic effect is reproducible in longer and
larger studies as
described above, it is hypothesized that the differences in bone formation and
resorption between
PTH and PTHrP also may result from their different pharmacokinetics following
subcutaneous
absorption as described above. It is well known that longer exposure of
osteoblasts or their
precursors in vitro or in vivo to PTH diminishes the anabolic response,
whereas it augments the
osteoclastic resorptive response (see,
Rosen & Bilezikian, JClin Endocrinol Metab. 86: 957-964 (2001); Dempster et
al.,
Endocrine Reviews 14: 690-709 (1993); Dobnig & Turner, Endocrinology 138: 4607-
4612
(1997)). By serendipity, the accelerated absorption and clearance of PTHrP
following
subcutaneous injection, as compared to those of PTH, may further favor the
formation vs.
resorption balance.
The doses of PTHrP employed in this study were very large. Subjects in this
study
received an average dosage 410.25 g per day in the eight subjects who
received PTHrP. This is
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some 10- to 20-fold larger than doses of hPTH(1-34) (20-40 g/day) commonly
employed in
osteoporosis studies. Doses of PTH in excess of 20 gg/day are known to be
associated with
hypercalcemia and other adverse effects. It is surprising, therefore, that
healthy subjects would
tolerate PTHrP doses of this magnitude without developing hypercalcemia,
postural hypotension,
nausea, flushing or other adverse effects. The differences cannot be ascribed
to differences in
molar amounts of the two peptides employed, for PTHrP(1-36) is very close in
molecular weight
to PTH(1-34) (approximately 4200 Mr). Nor can the differences be ascribed to
different
interactions with the common PTH/PTHrP receptor: both hPTH(1-34) and hPTHrP(l-
36) display
similar or identical binding kinetics and signal transduction activation
characteristics, in humans.
Different serum metabolic clearance rates are an unlikely explanation. as
well, for it has been
demonstrated that the T1i2 of intravenously infused PTHrP(1-36) is about six
minutes,
indistinguishable from the approximately five to six minutes reported for
hPTH(1-34). Without
being restricted to theory, one possible explanation is that the differences
in skeletal effects of
the two peptides relate to differing pharmacokinetic characteristics of PTH
and PTHrP following
subcutaneous injection. Human PTH(1-34) reaches peak plasma levels at about 30-
45 minutes
following injection, whereas peak plasma levels of PTHrP occur at or before
about 15 minutes
following a subcutaneous dose. Thus, hPTHrP(1-36) is likely more rapidly
absorbed than PTH
following subcutaneous injection, and plasma levels of PTHrP reach their peak
and decline more
rapidly than those of PTH.
These pharmacokinetic differences may also account for the selective or pure
anabolic
response observed. It is well known that longer exposure of osteoblasts in
vitro or in vivo to
PTH diminishes the anabolic response, whereas it augments the osteoclastic
resorptive response.
The accelerated absorption and clearance of PTHrP following subcutaneous
injection, as
compared to those of PTH, may further favor the formation vs. resorption
balance.
In this study, subjects in both the placebo and PTHrP groups were
concomitantly
receiving estrogen, in addition to calcium and vitamin D supplements, in part,
for ethical reasons,
so that the placebo group would receive some form of currently accepted
treatment for
osteoporosis. As for PTH, it remains to be determined whether the anabolic
effect of PTHrP is
enhanced by concomitant use of estrogen. Studies using PTH in humans in
general show similar
efficacy whether the subjects are receiving estrogen or not (see, FIG. 8),
although there have
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been no studies to date directly addressing this question for PTHrP. Whether
PTHrP might be
more or less effective when given concomitantly with other anti-resorptive
agents
(bisphosphonates, selective estrogen receptor modulators, etc.) remains to be
determined.
Short-term, very high dose treatment with PTHrP(1-36) causes a remarkable
increase in
spine BMD. In contrast to the combined or net resorptive and anabolic skeletal
effects of
intermittently administered PTH, PTHrP appears to have predominantly anabolic
effects with
little or no resorptive component. The differences between PTH and PTHrP are
not likely to
reflect differences in receptor interactions or signaling between the two
molecules, but likely
reside in the differing pharmacokinetic properties of the two molecules
following subcutaneous
administration.
Of the seven subjects receiving placebo for four months, six subjects
demonstrated no
significant change in bone mineral density (BMD) at either the hip or spine.
One placebo subject
did display a 6% increase in spine BMD. This is clearly not the expected or
typically
encountered response to placebo (The writing group for the PEPI trial, JAMA
276: 1389-1396
(1996); Delmas et al., NEngl JMed 337: 1641-1647 (1997); Chestnut et al.,
Osteoporosis Int 8
(suppl 3): 13 (1998); Liberman et al., NEngl JMed 333: 1437-1443 (1995);
McClung et al., N
Engl JMed 344: 333-40 (2001); Finkelstein et al., NEngl JMed 331: 1618-1623
(1994);
Hodsman et al., J Clin Endocrinol Metab 82: 620-28 (1997); Lindsay et al.,
Lancet 350:
550-555 (1997); Neer et al., NEngl.JMed 344: 1434-1441 (2001); Roe et al.,
Program and
Abstracts of the 81st Annual Meeting of the Endocrine Society, p. 59 (1999);
Lane et al., JClin
Invest 102: 1627-1633 (1998)), suggesting that this subject may have had
baseline vitamin D
deficiency, or an incidental radiologically non-apparent vertebral compression
fracture.
As illustrated in FIG. 4, the eight subjects receiving PTHrP demonstrated
important
increases in lumbar spine BMD, with a mean value of approximately 4.75%. When
compared to
all seven controls, including the placebo outlier, the results are significant
(p=0.026). When
compared to the six truly normal placebo controls, the results are highly
significant (p=0.003).
These results are quite extraordinary and surprising for several reasons.
First, none of the
available osteoporosis drugs, the anti-resorptives, yield these kinds of
increments in BMD in
such a short time frame (The writing group for the PEPI trial, JAMA 276: 1389-
1396 (1996);
Delmas et al., N Engl JMed 337: 1641-1647 (1997); Chestnut et al.,
Osteoporosis Int 8(suppl
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3): 13 (1998); Libennan et al., N Engl JMed 333: 1437-1443 (1995); McClung et
al., N Engl J
Med 344: 333-40 (2001)). As illustrated in FIG. 8, the rate of increase in BMD
observed in the
present study are greater than the rates of BMD increase reported in previous
clinical studies.
The results are extremely rapid: three months of PTHrP-(1-36) therapy yielded
increases not
generally observed for two to three years with anti-resorptives as described
above. Indeed,
several available anti-resorptives (SERMs, calcitonin, vitamin D, calcium)
never achieve these
increments in BMD.
Second, the results are comparable, or superior, to those achieved using PTH,
the best
studied anabolic skeletal agent to date (Finkelstein et al., NEngl JMed 331:
1618-1623 (1994);
Hodsman et al., J Clin Endocrinol Metab 82: 620-28 (1997); Lindsay et al.,
Lancet 350:
550-555 (1997); Neer et al., NEngl JMed 344: 1434-1441 (2001); Roe et al.,
Program and
Abstracts of the 81 st Annual Meeting of the Endocrine Society, p. 59 (1999);
Lane et al., J Clin
Invest 102: 1627-1633 (1998)).
Third, the doses required are surprisingly high: as noted earlier, standard
doses of PTH-
(1-34) are in the 20-40 g/day range (Finkelstein et al., NEngl JMed 331: 1618-
1623 (1994);
Hodsman et al., J Clin Endocrinol Metab 82: 620=28 (1997); Lindsay et al.,
Lancet 350:
550-555 (1997); Neer et al., NEngl JMed 344: 1434-1441 (2001); Roe et al.,
Program and
Abstracts of the 81st Annual Meeting of the Endocrine Society, p. 59 (1999);
Lane et al., J Clin
Invest 102: 1627-1633 (1998)), some 10-20-fold lower than those employed
herein for PTHrP-
(1-36).
Fourth, despite the relatively enormous doses of PTHrP administered in the
present study,
no adverse events have been encountered, whereas such adverse events have been
noted with far
smaller doses of PTH (Finkelstein et al., NEngl JMed 331: 1618-1623 (1994);
Hodsman et al.,
J Clin Endocrinol Metab 82: 620-28 (1997); Lindsay et al., Lancet 350: 550-555
(1997); Neer et
al., N Engl J Med 344: 1434-1441 (2001); Roe et al., Program and Abstracts of
the 81 st Annual
Meeting of the Endocrine Society, p. 59 (1999); Lane et al., J Clin Invest
102: 1627-1633
(1998)). The absence of toxicity and the requirement for high doses in humans
appears
comparable to the fmdings in rats described above, in which equimolar doses of
PTHrP had less
efficacy and less toxicity as compared to PTH. These observations, as noted
above, appear to
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reflect the serendipitous and non-predictable differences in pharmacokinetics
of PTHrP as
compared to PTH following subcutaneous administration.
Fifth, PTHrP is widely viewed as the quintessential catabolic skeletal hormone
responsible for dramatic skeletal mineral losses in patients with HHM. The
observation that
PTHrP is actually markedly anabolic for the skeleton when administered
"intermittently" (e.g.,
once per day) was not anticipated. This is evidenced by the fact that many
investigators and
pharmaceutical finns have worked for more than 10 years (and likely as long as
70 years) with
PTH in osteoporosis, but none has embraced PTHrP despite its having been in
the public domain
since its initial description in 1987.
Finally, the treatment regimen of the present invention for the treatment of
osteoporosis
has one additional unanticipated and unpredictable strength relating to
safety. In preclinical
toxicity studies, PTH was administered to growing rats for two years. Some
rats developed
osteosarcomas after approximately one year of PTH therapy. This suggests that
anabolic agent
use for periods of less than one year may put humans at less risk than those
used for longer
periods of time. The early efficacy of PTHrP in human studies suggests that
briefer durations of
treatment are likely to be effective in humans. Supporting this is the
observation that despite the
very high doses of PTHrP employed in this study, adverse events have not been
observed in
human subjects. In addition, the availability of a purely or predominantly
anabolic agent may
permit combined approaches to treating osteoporosis using concomitant,
intermittent or
sequential regimens with anti-resorptive agents. According to the methods of
the present
invention, patients can be treated, for example, initially with a several
month course of PTHrP,
or an analog or fragment thereof, and then switched to an oral anti-resorptive
formulation with
no osteosarcoma risk.
In summary, this short-term, high dose treatment with PTHrP(1-36) causes a
remarkable
increase in spine B1VID. Similar studies with even higher doses, e.g., 1000 to
3000
micrograms/day show comparable increases in bone density with no observable
adverse effects.
In contrast to the combined or net resorptive and anabolic skeletal effects of
intermittently
administered PTH over the same time period, PTHrP may have predominantly
anabolic effects
with little of a resorptive component. The differences between PTH and PTHrP
are not likely to
reflect differences in receptor interactions or signaling between the two
molecules, but likely
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reside in the differing pharmacokinetic properties of the two molecules
following subcutaneous
administration. Despite the very high doses of PTHrP employed, adverse events
have not been
observed in-18 human subjects. The availability of a purely or predominantly
anabolic agek,-'in
addition to PTH, may permit additional combined approaches to treating
osteoporosis using
concomitant, intermittent or sequential'regirnens'witli anti-iresorptive
agents.
EXAMPLE 2 CHARACTERIZATION OF PTHRP ANALOGS USING HUlVIAN
BONE AND RENAL RECEPTORS
The purpose of the present study was to characterize various-PTH- ancl--PTHrP-
anaiogs-
using human bone and human renal receptors. The ability of these analogs to
stimulate adenylate
cyclase was also examined. For a detailed description of the methods in the
present example, see
e.g., Orloff et al. Endocrinol., 131: 1603-1611 (1992), incorporated herein by
reference.
Materials and Methods
Peptides
(Tyr36)hPTHrP-( 1-36)amide [hPTHrP-(1-36)], hPTHrP-(1-74), and hPTHrP-(37-74)
were prepared by solid phase synthesis as previously described (Orloff et al.
J. Biol. Chem., 131:
1603-1611 (1992); Stewart et al. J. Clin. Invest., 81: 596-600 (1988)).
Synthetic hPTH-(1-34),
(Nle8 18, Tyr34)hPTH-(1-34), bovine (b)PTH-(1-34), rat (r)PTH-(1-34), hPTHrP-
(1-86),
(Nle8 18,Tyr34) bPTH-(3-34)amide, (D-Trp12, Tyr34)bPTH-(7-34)amide,
(Tyr34)bPTH-
(7-34)amide, hPTHrP-(7-34)amide, and hPTH-(13-34) were purchased from Bachem,
Inc.
(Torrance, CA). bPTH-(1-84) was obtained from the National Hormone and
Pituitary Program
through the National Institute of Diabetes and Digestive and Kidney Diseases
(1vIDDK). (Tyr36)
chicken (c)PTHrP-(1-36)amide was purchased from Peninsula Laboratories, Inc.,
Belmont, CA.
hPTHrP-(1-141) was provided by Genentech, Inc., So. San Francisco, CA, and
transaminated
rPTH-(1-34) was provided by Dr. David L. Carnes, Jr. (San Antonio, TX).
Chicken PTH-
(1-34)amide, [Nle8 t$, D-Trp12]bPTH-(7-18)-hPTHrP-(19-34)NH2 and [D-
Trp12]hPTHrP-(7-18)
[Tyr34]bPTH-(19-34)NH2 were prepared by solid phase synthesis as described
(Caufield et al.
Endocrinol 123: 2949-2951 (1988); Chorev et al. JBone Min Res 4: S270 (1989)).
The peptide
concentration for all peptides used is given as the value detennined by amino
acid analysis. The
same batches of peptides were used in all studies.
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Radioioaination
Radioiodination of hPTHrP-(1-36) was performed using a modification of the
lactoperoxidase method as previously described (Orloff et al. J. Biol. Chem.,
264: 6097-6103
(1989); Orloff et al. JBone Min Res 6: 279-287 (1991)). Purification of
radioligand was
accomplished by reverse-phase HPLC using a 30 cm -Bondapak C 18 coTumn-
(Waters
Associates, Milford, MA). The radioligand prepared and purified in this manner
is composed
almost exclusively of the monoiodinated form. The specific activity ranged
from 300-450
Ci/ g at the time of iodination. The radioligand displayed full biological
activity in the canine
renal adenylate cyclase assay when compared to the unlabeled peptide (Orloff
et al. J. Biol.
Chem., 264: 6097-6103 (1989)).
Cell culture
The human osteoblast-like osteosarcoma cell line, SaOS-2 (American Type
Culture
Collection, Rockville, MD), was maiintained in McCoy's medium supplemented
with 10% fetal
bovine serum, 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (50
g/ml). The
medium was changed every other day, and studies were performed at 5-7 days
post confluence.
Cell numbers were determined using a Coulter counter.
Preparation of membranes
Highly purified human RCM were prepared using discontinuous sucrose gradient
ultracentrifugation as previously described (Orloff et al. JBone Min Res 6:
279-287 (1991)). All
steps were performed in the presence of the following protease inhibitors:
aprotinin [10
Kallikrein inhibitor units '(KIU/ml], pepstatin (5 g/ml), leupeptin (45
g/ml), and
phenylmethanesulfanylfluoride (10 g/ml). Normal human kidney cortex was from
four separate
nephrectomy specimens removed for localized transitional cell carcinoma, renal
cell carcinoma,
or benign cysts. Renal function in all individuals was normal as assessed by
serum creatinine
and pyelography. Membranes were pooled, aliquoted, and stored at -70 C for
later use.
SaOS-2 cell membranes were prepared as previously described in detailr
Briefly,
postconfluent cells in 150 cm2 flasks were scraped into membrane buffer [10 mM
Tris HCl (pH
7.5), 0.2 mM MgC12, 0.5 mM EGTA, 1 mM dithiothreitol, leupeptin 45 g/ml,
pepstatin 5 g/ml,
aprotinin 10 KIU/ml, and phenylmethane sulfanyl-fluoride 10 g/ml] at 0 C.
Cell disruption was
achieved by sonification and the suspension was centrifuged at 13,000 x g for
15 min at 4 C.
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The pellet was resuspendbd"witYi"4"19duited glass homogenizer in membrane
buffer (minus
dithiothreitol) containing 250 mM sucrose. The suspension was layered onto a
cushion of the
membrane buffer containing 45% sucrose and centrifuged at 70,000 x g for 30
min at 4 C. The
membrane fraction layering at the interface was collected, diluted 5-fold with
membrane buffer
containing 250 mM sucrose, and recentrifuged. The pellet was resuspended in
membrane buffer
containing 250 mM sucrose, aliquoted, and stored at -70 C. Protein
concentrations were
determined by the method of Lowry using BSA as standard.
Receptor binding studies
The membrane binding assay utilizing human RCM at 30 C has been described
previously (Orloff et al. J Bone Min Res 6: 279-287 (1991)). Human RCM were
.added to a final
concentration of 90 g/ml. Total binding (TB) of 125I-(Tyr36)hPTHrP-(1-36)NH2
to human RCM
varied between 11 % and 20% of total counts added and nonspecific binding
(NSB) ranged from
2.4-4.0%. Specific binding of 125I-(Tyr36)hPTHrP-(1-36)NH2 reached equilibrium
by 30 min at
30 C. The incubation time of 30 min was used for subsequent equilibrium
binding competition
studies.
The binding assay for SaOS-2 membranes was conducted as for human RCM.
Membranes were added to a final concentration of 112.5 g/ml and specific
binding reached
equilibrium by 60 min at 30 C. TB ranged from 15-20% and NSB from 4.0-4.3%.
Binding to intact SaOS-2 cells was performed as described (Orloff et al. Am
JPhysiol
262: E599-E607 (1992)) with the following modifications. Binding studies were
conducted at
15 C in the presence of chymostatin (100 g/ml) and bacitracin (200 g/ml).
Specific binding of
125I-(Tyr36)hPTHrP-(1-36)NH2 reached equilibrium by 150 min at 15 C. The
incubation time of
150 min was therefore used for competitive binding studies. Cell viability, as
assessed by
exclusion of trypan blue, was greater than 95% at the end of a standard
incubation. Total
binding (TB) ranged from 18-23% of total radioactivity added and non-specific
binding (NSB)
consistently ranged between 5-7%.
Stability of radioligand during incubation under respective assay conditions
for each
membrane preparation (human kidney and SaOS-2 membranes) and for the intact
cell assay
(SaOS-2) was examined by the ability of 125I-(Tyr36)hPTHrP-(1-36) exposed to
cells to rebind as
compared to binding of "fresh" radioligand (Orloff et al., JBiol Chem 264:6097-
6103, (1989);
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. 6: ~79=287 ~1~91)). Specific rebinding of 125I-(Tyr3 ) hPTHrP-
Orloff et al. JBone Min Res .
(1-36) to human RCM, SaOS-2 membranes, and SaOS-2 intact cells was 92%, 98%,
and 83%
respectively. This indicated that significant degradation of radioligand did
not occur under the
respective assay conditions.
Adenylate cyclase assay
Adenylate cyclase-stimulating activity was examined in confluent SAOS-2 cells
as
previously described for ROS 17/2.8 cells (Merendino et al., Science 231:388-
390, (1986)), with
the following modification. The intact cell assay was conducted at 15 C, the
same conditions
employed for binding to intact SaOS-2 cells (vide supra). Time course
experiments
demonstrated that peak cAMP stimulation for PTHrP and PTH occurred after a 60
min
incubation. Dose response curves for each peptide were thus generated using 60
min incubations
at 15 C under binding assay conditions. Under these conditions, maximal
stimulation varied
between 80- and 200-fold above basal activity.
Adenylate cyclase activity was examined in human kidney membranes and SaOS-2
cell
membranes as previously described in detail for canine renal membranes (Orloff
et aL, J Biol
Chem 264:6097-6103, (1989); Orloff et al. JBone Min Res 6: 279-287 (1991)),
with the
following modifications: Time course experiments conducted at 30 C
demonstrated peak cAMP
accumulation at 10 min for human kidney membranes and 30 min for SaOS-2
membranes.
Therefore, dose response curves for each peptide were generated at 30 C for 10
min in human
kidney and 30 C for 30 min in SaOS-2 membranes. As with the intact cell
adenylate cyclase
assays, kidney and bone cell membrane adenlyate cyclase assays were performed
under binding
assay conditions. Results are expresses as percentage of maximal cAMP
stimulation in order to
compare peptide dose responses from different experiments. Maximal
cAMP.stimulation varied
from 3- to 8-fold above basal for human RCM, and from 2- to 7-fold for SaOS-2
membranes.
Data analysis
IC50 values for competitive binding experiments and EC50 values for adenylate
cyclase
dose response curves were determined from the concentration of peptide
yielding 50% of the
maximal response. Statistical differences were assessed by paired and unpaired
two-tailed
Student's t test. Further analysis of competition binding data was carried out
with the LIGAND
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computerized least-squares nonlinear curve-titting program (Munson et al. Anal
Biochem 107:
220-239 (1980)).
Results
Binding Studies
Competitive binding data using 125I-hPTHrP-(1-36) as radioligand in each of
the three
tissue preparations is shown in FIG. 9 and Table II (below). Binding of
radioligand was
completely displaced by all PTH and PTHrP analogs in each tissue examined,
except for
hPTHrP-(37-74), which, as expected, did not inhibit binding of 125I-hPTHrP-(1-
36). Scatchard
analysis of the data (FIG. 9, bottom panels) with the LIGAND computer program
was
compatible with a single class of high-affinity receptor sites in each tissue.
Receptor numbers,
calculated from the B,,,a,, values, were 0.24 0.06 and 0.36 0.08 pmol/mg
membrane protein
for human RCM and SaOS-2 membranes, respectively, and 25,900 1500 receptors
per cell for
SaOS-2 intact cells.
Competition of radiolabeled PTHrP binding with PTH and PTHrP agonists was
first
compared in RCM and SaOS-2 membranes (Table II and FIG. 9, Panels A and B). In
general,
the relative affinity of selected agonists in RCM closely paralleled that
observed in SaOS-2
membranes. rPTH-(1-34), bPTH-(1-34), and cPTHrP-(1-36) displayed similar
relative affinities
as compared to hPTHrP-(1,36), while (N1e8 18Tyr3~)hPTH-(1-34) and cPTH-(1-
34)NH2 were less
potent than hPTHrP-(1-36) in both assay systems. The relative affmity of bPTH-
(1-84) was.
approximately 10-fold less than the amino-terminal analogs. Overall, these
studies disclosed no
important differences between PTH/PTHrP binding in bone as compared to kidney.
Adenylate Cyclase Assay
The relative affinity of the agonist analogs in the binding assays was
reflected in their
adenylate cyclase-stimulating potency, with two notable exceptions (Table II
and FIG. 10).
Although rPTH-(1-34) was similar in binding affinity to hPTHrP-(1-36) in RCM
and SaOS-2
membranes, it was 10-fold more potent in stimulating adenylate cyclase in both
membrane
preparations. bPTH-(1-84), which displayed lower binding affinity, retained
its lower relative
potency as compared to hPTHrP-(1-36) in. the SaOS-2 membrane adenylate cyclase
assay, but it
was essentially equipotent to hPTHrP-(1-36) in stimulating cAMP production in
RCM.
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In order to investigate whether differences existed between intact and broken
cell
preparations, SaOS-2 intact cells were also studied (Table III and FIGS. 9C
and lOC). In
general, the relative affinity and cAMP-stimulating potency of the peptide
agonists that were
tested closely paralleled the results in RCM and SaOS-2 membranes. However,
the absolute
potency for some of the amino-terminal analogs varied between 2- to 4-fold
less than that
observed in either SaOS-2 membranes or RCM. Interestingly, rPTH-(1-34) did not
demonstrate
enhanced second messenger coupling relative to its binding affinity in SaOS-2
cells (Table III),_ a
pattern which had been observed in RCM and SaOS-2 membranes (Table II). The
affinity of
hPTHrP-(1-74) was substantially less than that of hPTHrP-(1-36), although this
difference was
greater for RCM (25-fold) than for SaOS-2 cells (9-fold). Interestingly,
hPTHrP-(1-141) had
5-fold greater affinity than hPTHrP-(1=74) in both assays, but it remained
less potent than
hPTHrP-(1-36). The'relative affinity of bPTH-(1-84) was similar to that of
hPTHrP-(1-74), but
as noted in the preceding paragraph, it did not display the enhanced coupling
to adenylate
cyclase in SaOS-2 cells or membranes as it had in RCM.
EXAMPLE 3 CHARACTERIZATION OF PTHRP ANALOGS USING CANINE
RENAL RECEPTORS
The purpose of the present study was to compare the properties of renal
receptors for
PTH and PTHrP and determine if the two peptides interact with the same
receptors. To
accomplish this aim, the PTH-related peptide, [Tyr36]PTHrP-(1-36)amide (PTHrP-
(1-36)), and
[NleB'18,Tyr34]hPTH-(1-34)amide (NNT-hPTH-(l-34)) were radioiodinated and used
in
competition binding studies using canine renal cortical membranes (CRMC) to
assess the
binding of several PTH and PTHrP analogs. The ability of these PTH and PTHrP
analogs to
stimulate adenylate cyclase was also examined. For a detailed description of
the methods in the
present example, see e.g., Orloff et al. J. Biol. Chem., 264: 6097-6103
(1989), incorporated
herein by reference.
Materials and Methods
Peptides
The PTH-related peptide (Tyr36) PTHrP-(1-36)amide (PTHrP-(1-36)) was prepared
by
solid-phase synthesis as previously described (Stewart et al., J. Clin.
Invest., 81: 596-600
(1988)). PTHrP-(49-74) and (CysS,Trp11,Gly13)PTHrP-(5-18) (Pl-peptide) were
prepared using
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similar solid-phase methods. Synthetic [rTle" 1",Tyr34]hPTH-(1-34)amide (NNT-
hPTH-(1-34))
and bovine PTH (bPTH)(1-34) were purchased from Bachem Inc., Torrance, CA. The
peptide
concentration for all peptides used is given as the value determined by amino
acid analysis and
not as the dry weight of the peptide.
Radio-iodination
Radio-iodination of the peptides PTHrP (1-36) and NHT-hPTH (1-34) was
performed
using a modification (Thorell et al., Biochim. Biophys. Acta, 251: 363-369
(1971)) of the
lactoperoxidase method (Marchalonis, Biochem. J., 113: 299-305 (1969)). The
peptide
(10 g/10 1) was mixed with Na 1251 (1 mCi/10 1) (Amersham, Arlington
Heights, IL) and
, lactoperoxidase (2 g) (Sigma Chemicals; St. Louis, MO)... The reaction was
initiated by the ..
addition of hydrogen peroxide (20 l of 0.03% H202) and was maintained by
three further 20 1
at additions of 0.03% H202 at 2.5 mm intervals for a total of 10 min. The
iodination mixture was
then applied to a C18 Sep-Pak cartridge (Waters Associates, Milford, MA). The
cartridge was
washed with 3 ml 0.1% TFA, and then eluted with 3 m175:25% acetonitrile: H20
(v:v)
containing 0.1% TFA into borosilicate glass test tubes containing 30 l of 2%
BSA. The eluate
was lyophilized and purified by reverse-phase HPLC using a 30 cm u-Bondapak
C18 column
(Waters Associates). The column was equilibrated with H20 containing 0.1% TFA
and
developed with acetonitrile in 0.1% TFA. For 1251 NNT-PTH (1-34), the gradient
employed was
a 60 min linear gradient of 33-43% acetonitrile. For 125I PTHrP-(1-36),
elution was
accomplished with a 50 min linear 27-34% acetonitrile gradient. Eluted
fractions were collected
in borosilicate glass tubes (12 x 75mm) containing 30 l of 1% BSA and
monitored for
radioactivity in a gamma spectrometer.
Analysis of Radioligand
HPLC-purified radioligand was subjected to complete enzymatic digestion in 100
l of a
buffer consisting of Tris-HC1(50 mM) pH 7.5, NaCI (75 mM), and sodium aside
(0.005%)
(Brown et al., Biochem., 20: 4538-4546 (1981)). A mixture of trypsin (1 g/10
l),
carboxypeptidase Y (1 g/10 l), leucine aminopeptidase (1 g/10 1), and
pronase E
(2 g/10 l) (all from Sigma, St. Louis, MO) was added and digestion carried
out at 37 C for 24
hours. The reaction was stopped by adding 100 l of 0.1% TFA. A 100 l aliquot
of the digest
was injected, along with 2 nmol each of nonoiodotyrosine and diiodotyrosine
standard, onto a
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C18 u-Bondapak column. The column was eluted with a linear gradient of 15-30%
methanol in
0.1% TFA over 30 mm at a flow rate of 1.5 ml/mm, and fractions (600 l) were
counted. UV
absorbance at 214 nm was monitored.
Preparation of Membranes
Highly purified canine renal cortical membranes (CRCM) were prepared using-a
modification of the procedure of Fitzpatrick et al. (J. Biol. Chenz., 244:
3561-3569 (1969)). The
renal cortex from adult mongrel dogs was homogenized in 3 volumes (ml:gm) of
0.25 M sucrose
containing 5.0 mM Tris HCl (pH 7.5), 1.0 mM EDTA, 6.5 KIU/ml aprotinin and 50
g/ml
bacitracin (SET buffer) at 4 C with ten 30 second strokes of a motor-driven
teflon pestle at 2000
RPM. The homogenate was filtered through one thickness of nylon mesh and
centrifuged at
1475xg for 10 mm. The supernatant was discarded and the pellet resuspended in
1 volume of 2.0
M sucrose, 5 mM Tris HC1, 1 mM EDTA (pH 7.5), 6.5 KIU/ml aprotinin, and 50
g/ml
bacitracin. This was centrifuged at 13,300xg for 10 minutes and the pellet
discarded. The
supematant was diluted 8-fold with ET buffer (5 mM Tris HCI, 1 mM EDTA (Ph
7.5), 6.5
KIU/ml aprotinin, and 50 g/ml bacitracin) and centrifuged at 20,000xg for 15
min. The
supematant was discarded and the white upper layer of the pellet removed and
resuspended in
one volume of SET buffer. The 20,000xg centrifugation was repeated two more
times, and the
white pellet suspended in one volume of SET buffer. These are referre=d to as
"crude CRCM."
Membranes were purified further by a modification of the procedure described
by Segre
et al. (J. Biol. Chem., 254: 6980-6986 (1979)). The white pellet described
above was
centrifuged at 2200xg for 15 mm and the supernatant and upper portion of the
resulting double-
layered pellet was removed and resuspended in SET buffer. This was centrifuged
at 20,000xg for
15 mm and the supernatant discarded. The pellet was then layered onto a
discontinuous gradient
of sucrose in 0.01 M Tris, 0.001 M Na2EDTA (pH 7.5), 6.5 KIU/ml aprotinin, and
50 g/ml
bacitracin. The gradient consisted of 39% sucrose (2 ml), 37% sucrose (4 ml),
and 32% sucrose
(2 ml). The membranes were centrifuged at 25,000 rpm (75,000xg) for 90 mm at 4
C. Major
bands were present at each interface in addition to a pellet at the bottom of
the tube. Preliminary
studies of the lightest fraction (not entering the sucrose) and the fraction
at the 32%-37%
interface indicated the highest specific binding and lowest non-specific
binding. The lightest
fraction, however, demonstrated less degradation of the radioligand in
rebinding studies.
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Therefore, all subsequent experunents were coriducted with this fraction
except where
specifically indicated.
The above membranes were diluted with three volumes of ET buffer, centrifuged
for
15 min at 7,800xg, suspended in one volume SET, aliquoted into 750 i aliquots
and stored at
-70 C. Membranes so prepared retained full receptor binding activity for at
least a 6-month
storage period. A single membrane preparation was used for all conventional
binding
experiments.
A second membrane preparation was performed using the same procedure as above;
but
in the presence of leupeptin (5 g/ml), pepstatin (5 gg/ml), aprotinin (10
KItJ/ml),
N-ethylmaleimide (NEM) (1.0 mM), and phenylmethanesulfanyl fluoride (PMSF) (10
g/ml) in
all steps (Nissenson et al., Biochem., 26: 1874-1-878 (1987)). -Protein was-
measured by the
method of Lowry using BSA as standard.
Receptor Binding Studies
Binding assays were conducted in siliconized 12x75 mm borosilicate glass test
tubes at
20 C in a final volume of 0.2 ml. The binding buffer consisted of 50 mM Tris
HC1 (pH 7.5),
4.2 mM MgC1a, 0.3% BSA, 26 mM KCI, approximately 60-80x103 cpm/tube of
radioligand, and,
where appropriate, unlabeled peptides. Based on radioligand stability studies
described below,.
bacitracin was added to a fmal concentration of 100 gg/ml for experiments
conducted with
121l NNT-hPTH-(1-34) and 200 g/ml for 125I PTHrP-(1-36). Binding was
initiated by adding
50 g membrane. At the end of the incubation periods described, 50 l
triplicate aliquots were
layered onto 300 l of iced binding buffer containing 1.0% BSA in 500 i
polypropylene tubes.
The tubes were centrifuged at approximately 16,000xg for three min at 4 C in a
microcentrifuge.
The supernatant was aspirated arid the tip of the tube containing the membrane-
associated
radioligand was cut off. Radioactivity in both the pellet and supernatant was
measured.
Total binding (TB) of radioligand varied between 7.2-14.6% of total counts
added for
121I NNT-hPTH-(1-34) and 25.5-30.0% for 1251 PTHrP-(1-36). Nonspecific binding
(NSB) was
1.8 ,0.3% ( SEM) for 125I NNT-hPTH-(1-34) and 9.9 0.8% for 125I PTHrP-(1-
36). Recovery
of both radioligands from incubation and wash tubes was routinely in excess of
95%.
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Adenylate Cyclase Assay
Adenylate cyclase-stimulating activity was examined using a guanyl nucleotide-
amplified
canine renal cortical membrane (CRCM) PTH-sensitive adenylate cyclase assay,
performed as
previously described in detail (Stewart et al., Proc. Natl. Acad. Sci. USA,
80: 1454-1478 (1987)).
Briefly, synthetic PTHrP-(1-36) or bPTH-(1-34) was added in duplicate to assay
tubes containing
crude CRCM, and the conversion of a-[32P]cAMP to [32P]cAMP at 30 C for 30 min
was
examined. Results are expressed as the percent increment in adenylate cyclase
activity in tubes
containing the peptides as compared with tubes containing vehicle only.
Adenylate cyclase-stimulating activity of both peptides was also examined
using highly
purified 32% interface membranes. Incubation was carried out under binding
conditions at 20 C
for 20 min in the presence of the protease inhibitor, bacitracin (200 gg/ml).
All other aspects of
this assay were identical to the standard assay.
Data Analysis
Dissociation constants (Kd) were determined by Scatchard analysis of the data
obtained
from competitive binding experiments using radioligand and increasing
concentrations of
unlabeled ligand. In competition studies using an unlabeled competitor
different from the
radioligand, binding affinities (Ki) were derived from the IC50 (concentration
of unlabeled ligand
displacing 50% of specific radioligand binding) using the computer program
EBDA (McPherson,
KINETIC, EBDA, LIGAND, LOWRY: A COLLECTION OF RADIOLIGAND BINDING ANALYSIS
PROGRAMS, pp. 14-97, Elsevier, Amsterdam (1985)). Statistical differences were
assessed by
paired Student's t test. Further analysis of competition curves was carried
out with the LIGAND
computerized least squares nonlinear curve-fitting program of Munson and
Rodbard (Anal.
Biochem., 107: 220-239 (1980)), modified for microcomputer use by McPherson
(Ibid.).
Computer fits of a one- or two-binding site model were compared, indicating
the statistically
preferred model. Significance was determined using a partial F-test.
Results
Chacterization of Ligand Binding; Association
Specific binding of 125I NNT-hPTH-(1-34) reached equilibrium of 20 min at 20 C
(FIG. 11). Nonspecfic binding became relatively content by a 5 min at 2.5
0.1% (SEM) -of
total radioactivity added. For all subsequent equilibrium experiments, the
incubation time was
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20 min. Specific binding under these conditions ranged from 65-85% of total
bound
radioactivity for 125I NNT-hPTH-(1-34) and 55-75% of the total binding of 1211
PTHrP(1-36)
Binding Studies
Inhibition of binding of 125I-[Nle8'1$,Tyr34]hPTH-(1-34) amide was performed
using
increasing concentrations of unlabeled [Nle8'18,Tyr34]hPTH-(l-34)amide, bPTH-
(1-34), and
PTHrP-(1-36) under equilibrium conditions (FIG. 12). The PTH analogues were
slightly more
potent than PTHrP-(l-36) in inhibiting binding (less than 2-fold) with a mean
K; of 7.5 nM for
[Nle8'18, Tyr34]hPTH-(1-34)amide and 6.1 nM for bPTH-(1-34). The binding
affinity constant
(K,) for PTHrP-(1-36) was 11.5 nM (Table II, top).
When'ZSI-PTHrP-(1-36) was used as the radioligand, all three synthetic
peptides were,
approximately equipotent in inhibiting binding (FIG. 13). Binding dissociation
constants for
[Nle$'18,Tyr34]hPTH-1-34)amide, bPTH-(1-34), and PTHrP-(1-36) were 8.5,10.5,
and 14.1 nM,
respectively (Table II, top). Both PTHrP-(49-74) and a synthetic 13-amino acid
bio-inactive
amino-terminal PTHrP (P1 peptide) failed to inhibit binding of 121I-PTHrP-(1-
36) to canine renal
membranes (FIG. 13).
Representative Scatchard plots of the equilibrium binding data are presented
in FIGS. 12
and 13. The B,,,,,, value for PTH analogue was 2.73 0.31 pmol/mg protein and
for PTHrP-
(1-36) was 5.08 0.56 pmol/mg protein. Analysis of both sets of data with the
LIGAND
program demonstrated a single class of high affmity receptor sites; the data
would not fit a
two-site model.
In summary, each unlabeled PTH/PTHrP analog reduced the binding of each
radioligand
to the same degree, suggesting that the PTH/PTHrP analogs are binding to a
similar or identical
receptor. Scatchard analysis indicated a homogeneous class of high affinity
receptor sites
without significant cooperative binding interactions. Biologically inactive
PTHrP fragments
failed to displace the radioligand. These data, demonstrating similar binding
affinities and B,,,aX
values for PTHrP and PTH analogs in canine renal membranes, have also been
observed in bone
derived cells (Juppner et al., J. Biol. Chem., 263: 8557-8560 (1988)), in
canine renal membranes
and UMR-106 osteosarcoma cells (Nissenson et aL, J. Biol. Chem., 263: 12866-
12871 (1988)).
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Adenylate Cyclase Assay:
In contrast to their similar affinities in the binding assay, bPTH-(1-34) was
substantially
more potent than PTHrP-(1-36) in the canine renal cortical adenylate cyclase
assay (Table II).
This relationship was seen in both the standard assay conditions (30 C for 30
min) and under
binding assay conditions (20 C for 20 min, with bacitracin). In the standard
assay (30 min,
30 C), bPTH-(1-34) had greater than 6-fold the potency of PTHrP-(1-36) with K.
values of 0.06
and 0.40 nM, respectively. To exclude the possibility that selective
destruction of PTHrP
occurred during the assay in the presence of renal membranes, the adenylate
cyclase assay was
performed under binding conditions which had been demonstrated to result in
negligible
proteolysis of radioligands. Under conditions identical to the equilibrium
binding assay (20 C,
min, with bacitracin), adenylate cyclase stimulation by bPTH-(1-34) was 15-
fold greater than
for PTHrP-(1-36). The Kn, values under binding assay conditions were 0.13 and
2.00 nM,
respectively.
EXAMPLE 4 CHARACTERIZATION OF PTHRP ANALOGS USING
15 OVARIECTOMIZED, OSTEOPENIC RATS
Candidate PTHrP analogs are evaluated for their effect on bone mass in
ovariectomized
rats, generally in accord with the procedures of Stewart et al., J. Bone Min
Res, 15: 1517-1525
(2000), incorporated by reference herein. In the present Example, three
PTH/PTHrP molecules
were selected for direct comparison: PTH(1-34), PTHrP(1-36) and the PTH
analog,
20 SDZ-PTH-893 (LeuB, Asp10, Lysll, Ala16, Glnia, =Ibr33, Ala34hPTH(1-34)). A
six month study
was performed in which adult (six month old) vehicle-treated ovariectomized
(OVX) and sham
OVX rats were compared to OVX rats receiving 40 g/kg per day of either PTH(1-
34),
PTHrP(1-36) or PTH-SDZ-893.
Methods
Peptides and Peptide Administration
Recombinant human PTH(1-34) (rec hPTH(1-34) or LY333334) was prepared as
described previously (Hirano et al., JBone Min Res 14: 536-545 (1999); Frolick
et al., JBone
Min Res 14: 163-72 (1999)). PTHrP(1-36) was prepared using solid phase
synthesis as
described previously (Everhart-Caye et al., JClin Endocrinol Metab 81: 199-208
(1996); Henry
et al., J Clin Endocrinol Metab 82: 900-906 (1997); Plotkin et al., JClin
Endocrinol Metab 83:
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2786-2791 (1998)). The human and rat sequences of PTHrP(1-36) are identical.
SDZ-PTH-893
(LeuB, Asp10, Lys11, Ala16, Glnls; Thr33, A1a3~hPTH(1-34) (Gamse et al., JBone
Min Res
12(suppl): S317 (1997)) was prepared using solid phase synthesis. The mass
spectrum and
amino acid composition were determined to be correct for each peptide and
purity greater than
97% was confirmed by analytical reversed-phase HPLC. Peptides were
administered
subcutaneously in 0.OO1N HCl in saline containing 2% heat-inactivated
ovariectomized (OVX)
rat serum at pH 4.2.
Animals
All studies were performed using virus- and antibody-negative female Sprague-
Dawley
rats from Harlan Sprague-Dawley (Indianapolis, IN). All rats underwent sham
ovariectomy or
ovariectomy at 5 months of age. Studies began at six months of age, one month
following
ovariectomy or sham operation. Rats were maintained on a diet containing 0.5%
calcium and
0.4% phosphorus. The light cycle was 12 hours.
Protocol
The protocol employed is described in schematic form in Table IV. Animals were
randomly assigned to 17 groups of 10 as described in the Table. Except for
animals in the first
group which were sacrificed at five months of age, the remaining animals were
observed for one
month, and treatment with the various test peptides or vehicle was begun at
six months of age.
For the peptide-treated animals, the peptide was administered daily,
subcutaneously at a dose of
40 g/kg/day, in the vehicle described above. For vehicle-treated animals,
vehicle alone was
administered in an identical fashion.
Chemistries
Serum and urine chemistries as described in Table XI were performed using
standard
autoanalyser methods (Boehringer-Mannheim-Hitachi, Indianapolis, IN). Kidney
calcium
content was determined following extraction of whole kidneys in 5%
trichloroacetic acid,
followed by calcium measurement by calcium analyzer (Calcette, Midfield, MA).
Bone Mass Measurements
Bone mass was assessed using bone ash weight as well as DEXA measurements of
the
radius, femur and whole body. Whole body bone mineral content was determined
using a
Norland DXA Eclipse densitometer, and results are expressed in mg. Left femur
bone mineral
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density in mg/cm2 (BMD), bone mineral content in mg (BMC), and cross sectional
area in cm2
(X-area) were determined using a calibrated Hologic QDR 4500A densitometer
coupled to Small
Animal Regional High Resolution software, as performed by S. Orwoll at Oregon
Health
Sciences University, Portland OR. Left radius maximal length measurements were
performed
using Fowler/Sylvac Ultra-Cal III calipers (Newton, MA). Radius ash weight was
determined as
described (Hock et al., JBone Min Res 7.= 65-72 (1992); Hock et al.,
Endocrinology 125:
2022-2027 (1989)) following careful cleaning of the radius of non-skeletal
tissue, dehydration in
ether for 48 hours, followed by air drying for 24 hours, and ashing in a
muffle furnace
(Barnstead/Thermodyne, Dubuque, IA) at 850 C for 16 hours. Ash weights were
recorded in mg
using a microbalance.
Bone Histomorphometry
Bone histomorphometry was performed on methyl methacrylate embedded sections
of
the right tibia of each animal following sacrifice as described in Table IV.
Animals were labeled
using calcein, 30 mg/kg, administered subcutaneously seven and three days
prior to sacrifice.
Standard histomorphometric measures were performed as shown in Tables IV-VI
(Parfitt et al., J
Bone Min Res 2: 595-610 (1987)).
Biomechanical Measures of Strength
Three-point bending on the femoral mid-shaft and compression of the L5
vertebral body
were done at 37 C. Shearing of the femoral neck was done at room temperature.
Complete
methods for these tests have been reported previously (Sato et al.,
Endocrinology 138:
4330-4337 (1997); Turner and Burr, Bone 14: 595-608 (1993); Sato et al.,
Endocrinology 139:
4642-51 (1998), each incorporated herein by reference).
Statistical Analysis
Statistical analyses were performed using SAS software. Two-way analysis of
variance
was performed to determine if there were significant interactions between
treatments and time,
and if there were differences between agents. Pair-wise comparisons were done
by contrast
T-tests if significant interactions were present, and by Dunnett's test if no
significant interactions
were found. Level of significance was set at p < 0.05.
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Results
Statistically and quantitatively significant increases were observed in femur
cross-
sectional area, femoral bone mineral content, and bone mineral density of the
PTH, PTHrP and
SDZ-PTH groups, with a rank order of SDZ-PTH>PTH>PTHrP (see Table VIII).
Femoral bone
mineral content (FIG. 14) increased significantly and markedly in each of the
peptide-treated
groups, at each of the three time points assessed. No changes in femur length
were observed due
to treatment (see Table VIII).
There was no important difference in whole body BMC in the three peptide-
treated
groups as compared to their time-matched OVX controls (see Table VIII for
details). Radius ash
weight (see Table VIII) increased significantly during the study in the
peptide-treated animal
groups, increasing beyond the values obsezved in both the. OVX and sham
control groups.
Bone histomorphometry was performed in order to assess structural features of
the
skeletal changes as well as changes in bone turnover. As can be seen in FIGS.
15 and 16,
trabecular area (Tb.Ar) declined markedly in the OVX control animals and
remained depressed
throughout the study, as compared to the sham animals. In contrast, marked
increases in
trabecular area occurred in all three peptide-treated groups, with the same
rank order observed in
the bone mass measures: SDZ-PTH>PTH>PTHrP. The increased Tb.Ar in treated
animals was
principally the result of increased trabecular thickness, which resulted in
reduced trabecular
separation (see Table V).
Bone formation (MSBS) declined with age over the first 30 days in all animals
(FIG. 15;
see Table VI for greater detail). However, at each time point following
initiation of treatment,
bone formation parameters were significantly increased in all three peptide-
treated animal groups
as compared to the age-matched OVX and sham animals (Table VI, FIG. 15).
Bone resorption parameters declined with age in all five groups (FIG. 16; see
Table VII
for greater detail). In contrast to the differences in bone formation among
the groups, there were
no important differences in resorption parameters between OVX and sham animals
at any time
point.
Biomechanical measures improved in all three peptide treated groups (FIG. 17,
and see
Table IX and X for additional detail). At the lumbar spine, measures of
biomechanical strength
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increased with each of the peptides. At the femoral neck, ultimate load also
increased with all
three peptides. The changes were statistically significant and quantitatively
large. Importantly,
for all three peptides, biomechanical measures at the lumbar spine and femoral
neck exceeded
those found not only in the OVX controls, but also the sham controls.
At the mid-shaft of the femur, a cortical bone site, similar findings were
observed
(FIG. 17). In general, the three peptide-treated groups showed augmented or
improved
biomechanical parameters as compared to both the sham and OVX control groups,
and these
changes were statistically, quantitatively, and functionally very significant
(see Table X for
complete details).
Body weight increased with increasing age in all groups throughout the study
but there
were no significant differences among the treated and control,groups.-
Animalsgained weight at._.
approximately the same rate (see Table XI for details).
The mean serum calcium remained normal in the sham and OVX animals throughout
the
study (FIG. 18, see Table XI for complete details). This was true for the PTH-
and PTHrP-
treated animals as well. In contrast to these two treatment groups, frank
hypercalcemia occurred
in the SDZ-PTH-treated animals, with mean calcium concentrations of 11.3, 11.6
and 11.7 mg/dl
at months 1, 3 and 6, respectively. These differences were significant
statistically. They were
also significant biologically in that four of the 30 SDZ-PTH-893-treated
animals (13%) died
during the study at 75, 83, 130 and 133 days of treatment. While the mean
serum calcium was
normal in the PTH group, one PTH-treated animal (3%) died with hypercalcemia
at day 171. No
PTHrP-treated animals died during the study.
Discussion
Candidate PTHrP analogs, or other skeletal anabolic agents, can be tested
using the
methods described above. PTHrP analogs, or other skeletal anabolic agents,
useful in the
methods of the present invention are expected to significantly increase the
total bone calcium,
trabecular calcium, cortical bone calcium, trabecular thickness, and bone
volume over untreated
OVX controls.
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EXAMPLE 5 SYSTEM AND METHODS FOR DESIGN OF PEPTIDOMIMETICS
AND SMALL MOLECULES HAVING BIOLOGICAL ACTTVITY
SIMILAR TO PTHRP AND SKELETAL AN'ABOLIC AGENTS
As described above, PTHrP, PTH, and TIP peptides, as well as their receptors
and
resultant metabolic pathways, may be used to develop peptidomimetics and small
molecule
drugs, that are useful as agonists and antagonists of these skeletal anabolic
agents. As used
herein, a "peptidomimetic" refers to derivatives of the fragments or full
length peptides of the
skeletal anabolic agents PTHrP, PTH, or TIP, described above, that demonstrate
biological
activity involving modulation of bone mass, as well as mixtures,
pharmaceutical compositions,
and compositions comprising the same. A "small molecule drug" refers to a non-
naturally
occurring low-molecular weight compound, having similar activity. In either
case, the biological
activity of a peptidomimetic or small molecule drug can be agonistic or
antagonistic to that of
PTHrP, PTH, or TIP, or may include a spectrum of activity, i.e., may be
antagonistic to PTH
activity and agonistic to PTHrP activity.
As with PTH, the biological activity of PTHrP is associated with the N-
terminal portion,
with residues (1-30) minimally providing the biological activity. Truncated
forms of the 39
amino acid tubular infundibular peptide (TIP) are also being assayed for
biological activity.
Receptors for these agents are also targets for structural-based drug design.
As described
above, the 500-amino-acid PTH/PTHrP receptor (also known as the PTH1 receptor)
belongs to a
subfamily of GCPR that includes those for glucagon, secretin, and vasoactive
intestinal pepti-de.
The extracellular regions are involved in hormone binding, and the
intracellular domains, after
hormone activation, bind G protein subunits to transduce hormone signaling
into cellular
responses through stimulation of second messengers. These second messengers
likewise provide
drug targets.
Also described above, a second PTH receptor (PTH2 receptor) is expressed in
brain,
pancreas, and several other tissues. Its amino acid sequence and the pattern
of its binding and
stimulatory,response to PTH and PTHrP differ from those of the PTH1 receptor.
The
PTH/PTHrP receptor responds equivalently to PTH and PTHrP, whereas the PTH2
receptor
responds only to PTH. The endogenous ligand of this receptor appears to be
tubular infundibular
peptide-39 or TIP-39.
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In one aspect of the invention, these compositions modulate, i.e., upregulate
or
downregulate PTH1 or PTH2 receptor activity. In another aspect, a system
comprising structural
information relating to the atomic coordinates obtained b~ x-ray diffraction
of a PTHrP, PTH, or
TIP peptide, fragment, peptidomimetic or small molecule drug is provided. In
another
embodiment, and antibody to a PTHrP, PTH, or TIP peptide, fragment,
peptidomimetic or small
molecule drug is provided. In even another embodiment, a purified crystalline
preparation of a
PTHrP, PTH, or TIP peptide, fragment, peptidomimetic or small molecule drug is
provided.
Structures of PTH1 or PTH2 receptors, or a PTHrP, PTH, or TIP peptide,
fragment,
peptidomimetic or small molecule drug are obtained by x-ray diffraction of
crystallized
polypeptides, 2-D nuclear magnetic resonance spectroscopy of the same, or by
similar methods
of obtaining high resolution structures of biological materials. High
resolution structures refer to
structures solved to greater than 2.8 angstroms, and preferable greater than
2.3 angstroms, and
are used to map the active sites of these receptors and their ligands.
Structures are determined
and interpreted using computer systems described in the art, e.g., having at
least a memory bank,
a display, a data input means, a processor and an instruction set comprising
an algorithm for
reading, interpreting and rendering the structural data, all of which are well
known in the art, for
example see, U.S. Patent No.: 6,273,598 to Keck et al., entitled, Computer
system and methods
for producing morphogen analogs of human OP-1, incorporated herein by
reference. According
to the present invention, such systems may be standalone or networked, i.e.,
through a packet
switched network. Computer aided design (CAD) programs are employed to design
peptidomimetics and small molecule agents having the appropriate receptor
antagonist or agonist
activities, based upon the obtained structural maps. Candidate agents are
assayed for PTHrP,
PTH, and TIP-like biological activity using the assays described herein, as
well as similar assays
known in the art.
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TABLE I.
Baseline Demographics
PBO PTHRP p
(n=8) (n=8)
Age (years) 56.5 1:3 61.5 2.4 ns
Height (cm) 162.5 2.3 161.6 2.3 ns
Weight (kg) 62.1 2.7 62.3 3.0 ns
BMI 23.6 1.1 24.0 1.5 ,..ns
Plasma 25 D(nmol/L) 61.9 2.1 63.1 2.1 ns
Calcium intake (mg/day) 940 186 1438 296 ns
Yrs post Menopause 13.5 2.9 12.3 2.3 ns
Yrs on Estrogen 8.4 1.7 8.0 1.5 ns
# on Thyroxine 1 /8 0/8
Smoker 2/8 0/8
L/S BMD (gm/cm) 0.748 .03 0.763 .01 ns
L/S BMD (T-score) -2.71 .26 -2.58 .12 ns
T. Hip BMD (gm/cm2) 0.710 .02 0.722 .02 ns
T. Hip BMD (T-score) -1.9 .15 -1.77 021 ns
FN BMD (gm/cm2) 0.572 .02 0.654 .03 .05
FN BMD (T-score) -2.5 .21 -1.95 ,27 ns
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TABLE II
In vitro activity of [Tyr367 PTHrP-1-36) amide compared to bPTH-(1-34)
Kd I K, K.
Peptide 125 I-PTH I-PTHrP Standard Binding
assay conditions
nM
bPTH-(1-34) 6.1 1.5 10.5 4.4b 0.06 0.01 0.13 f 0.01
PTHrP-(1-36) 11.5 2.5 a 14.0 5.4b 0.40 0.07' 2.00 0.17
ap = 0.03.
Not significant.
'p<0.002.
Binding studies were conducted at 20 C using monoiodinated [Nle$''$'Tyr34]hPTH-
(1-34) amide (125I-PTH) or
Tyr36]PTHrP-(1-36)amide (125I-PTHrP) as the radioligand. The K~"values were
determined by Scatchard analysis,
and the K; values were derived from the IC50 values. Adenylate cyclase
stimulation was evaluated under standard
assay conditions, employing partially purified canine renal membranes and 30-
min incubations at 30 C. Adenylate
cyclase stimulation was also evaluated under binding assay conditions, using
highly purified canine renal
membranes in the presence of bacitracin (200 g/ml) and 20-min incubations at
20 C.
In vitro activity of PTH and PTHrP agonists in human RCM (kidney membranes)
as compared to SaOS-2 membranes
Binding (IC50) (nM) Adenylate cyclase (EC50) (nM)
Peptide Kidney SaOS Kidney SaOS
membranes membranes membranes membranes
(Tyr )hPTHrP-(1-36)NH2 0.42 0.07 0.64 Ø02 0.50 0.10 0.51 0.07
(Nlea'1sTyr34)hPTH-(1-34) 3.6 0.7 2.0 0.3 1.1 0.11 1.9 0.4'
bPTH-(1-34) 0.39 0.06 1.5 0.4 0.26 0.14 0.50 0.06
rPTH-(1-34) 0.35 0.15 0.56 0.06 0.05 0.016 0.09 0.036
cPTH-(1-34)NH2 21.5 8.5 a 20.0 5.01i 5.4 0.1 16.3 4.8a
(Tyr36)cPTHrP-(1-36)NH2 0.47 0.22 1.1 0.3 0.49 0.06 0.87 0.34
bPTH-(1-84) 5.1 2.3 b 8.0 2.0' 0.59 0.21 2.4 0.2d
Values are the mean SEM of two or more experiments for each peptide.
Statistical analysis vs.
(Tyr36)hPTHrP-(1-36)NH2:
"p<0.01.
b P < 0.05.
P < 0.001.
d P < 0.0001.
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'1'A1sLE III
In vitro activity of PTH and PTHrP agonists in human RCM (kidney membranes)
as compared to SaOS-2 intact cells
Binding (IC50) (nM) Adenylate cyclase (EC50) (nM)
Peptide Kidney SaOS cells Kidney SaOS cells
membranes membranes
[Tyr ] hPTHrP-(1-36) NH2 0.42 + 0.07 1.5 0.1 0.50 + 0.10 1.0 + 0.1
hPTH-(1-34) . 1.9+ 0.4" 3.1 +0.3b 0.70 + 0.40 1.6 + 0.0
[NleB'18, Tyr34] hPTH-(1-34) 3.6 + 0.7 2.8 + 0. 1' 1.1 + 0.1 2.3 + 0.4'
bPTH-(1-34) 0.39 0.06 1.3 0.1 0.26 + 0.14 1.2 + 0.1
rPTH-(1-34) 0.35+0.15 0.9+0.2' 0.05+0.010 0.9+0.11
cPTH-(1-34)NH2 21.5+8.5 0d 5.4+0.le 3.9+0.1'
[Tyr36] cPTHrP-(1-36) NH2 0.47 +_ 0.22 0d 0.49 +_ 0.06 0.8 + 0.1
hPTHrP-(1-74) 9.5+_3.5 -12.9+1.4b 7.8+0.8 9.2 1.0b
hPTHrP-(1-141) 2.0+0.1e""'" 2.4+0.1 1.3 0.4" 1.9+0.4e-
bPTH-(1-84) 5.1 + 2.3 c 17.5 + 2.5b 0.59 + 0.21 7.7 + 1.4b
Values are the mean + SEM of two or more experiments for each peptide.
Statistical analysis vs.
[Tyr36] hPTHrP-(1-36) NH2:
a P < 0.01.
b P < 0.0001.
P < 0.05.
d P 0, These peptides were tested in SaOS-2 membranes, not in SaOS-2 cells
(see Table II).
~ eP<0.001.
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O
TABLE IV
Protocol
Months of treatment
ID Surgery No/group
1 2 3 4 5 6 7
SB Sham 10 kill
VO OVX 10 kill
S30 Sham 10 hold vehicle
V30 OVX 10 hold vehicle Ln
P30 OVX 10 hold PTH N
R30 OVX 10 hold PTHrP
A30 OVX 10 hold STZ o
S90 Sham 10 hold vehicle vehicle vehicle
V90 OVX 10 hold vehicle vehicle vehicle W
P90 OVX 10 hold PTH PTH PTH IH
R90 OVX 10 hold PTHrP PTHrP PTHrP W
A90 OVX 10 hold STZ STZ STZ
S180 Sham 10 hold vehicle vehicle vehicle vehicle vehicle vehicle
V180 OVX 10 hold vehicle vehicle vehicle vehicle vehicle vehicle
P180 OVX 10 hold PTH PTH PTH PTH PTH PTH
R180 OVX 10 hold PTHrP PTHrP PTHrP PTHrP PTHrP PTHrP
A180 OVX 10 hold STZ STZ STZ STZ STZ STZ y
TABLE V
Structural Histomorphometry of the Right Proximal Tibia in Mature
Ovariectomized (OVX) Rats Given Once Daily rec hPTH-(1-34),
PTHrP-(1-36), or SDZ- PTH-893, for 30, 90, or 180 Days
Trabecular Area Trabecular Number Trabecular Space Trabecular Thickness
Tb.A. Tb.N. Tb.Sp. Tb.Th.
% #/min (Dm (Dm
Baseline OVX 17t2 3.1 +0.2 281 24 54t4
Baseline Sham 28 ~ 2 4.7 f 0.2 156 t 10 60 ~ 2
30 Days
Sham 20tla 3.9~0.2a 206 13 a 51 2 OVX 6 1 1.3+0.3 847 98 46f2 0
PTH(1-34) 19 2a 2.3 f0.1 a'c 367 ~ 28 a' 81 f 5 a' ~
PTHrP(1-36) 13 1abc 2.1 0.1 a 436 f 36 a 62 f 3abc N
SDZ PTH 893 23f3a 2.5f0.2a 348f42a 934a' CD
N
0
90 Days
Sham 20f2a 3.7+0.3a 230t27a 52f3 0w
OVX 3 f 1 0.6 f 0.1 2178 t 363 '51 6 W
PTH(1-34) 38 3' 2.4 0.2 8c 274 34 $ 158 f 6 a'c
PTHrP(1-36) 19 3a'b 2.1 0.2 a'c 398 f 35 8bc 89 f 5 8b
SDZ PTH 893 50f5a' 2.4f0.1a'c 210f23a 205f20a'c
180 Days =
Sham 15f3a 2.7 0.3a 374f78a 53 4 OVX 9 7 0.7f0.2 1923f492 77 32 y
PTH(1-34) 38 f 3ac 2.3 0.1 a 279 t 29 a 163 f 8 a'
PTHrP(1-36) 23 f 2a'bc 2.3 0.2 a 361 f 43 a 97 f 4ab'c
SDZ PTH 893 57 f 4 s'c 3.3 =1= 0.5 a b 139 =1= 26 a'b' 183 :L 20 $'c
Data are expressed a mean t SEM for 7 to 10 rats per group. Statistically
significant differences, p<0.05. Baseline data were not included in
statistical analyses, and are
shown for descriptive purposes,oniy. .
versus time-matched OVX
b versus time-matched PTH (1-34)
'versus time-matched sham
TABLE VI
Bone Forination Measures of the Right Proximal Tibia in Mature Ovariectomized
(OVX) Rats Given Once Daily
rec hPTH-(1-34) (LY333334), PTHrP-(1-36), or SDZ PTH 893, a PTHrP Analog, for
30, 90, or 180 Days
Mineralizing Apposition Rate Bone Formation Rate
Surface MSBS MAR BFR/BS
(%) ( m/d) (Fim/d)
Baseline OVX 37 f 1 4.7 f 0.8 1.77 f 27
Baseline Sham 32 t 2 4.4 f 0.4 1.42 20
30 Days
Sham 18f2a 2.8f0.la 0.55f6
OVX 26:L 2- 2.4t0.1 0.69f6
rhPTH(1-34) 49 f 2ac 2.4 f 0.1 1.18 f 7a
PTHrP(1-36) 39 f 1$'' 2.6 f 0.1 1.00 f 4abc o
SDZ PTH 893 56 3a 2.6 f 0.1 1.42 f 8a't''
90 Days o
Sham 14 l8 2.4 0.2 0.36f4
OVX 2112 2.4f0.1 0.51f6 w
rhPTH(1-34) 41 f la' 3.0 f 0.1a 1.23 48' IH
PTHrP(1-36) 37 11 a'b'c 3.6 f 0.7a 1.36 28a'b' W
SDZ PTH 893 44 f 2" 3.3 f 0.2a'0 1.45 f 10a6
180 Days
Sham 14+2 2.6f0.3 0.38 7
OVX 19t2 2.9 0.2 0.54f8
rhPTH(1-34) 40 f 2'~ 3.8 t 0.7 1.53 f 28a'
PTHrP(1-36) 29 l3.6 f 0.5c 1.05 f 13a'b'
SDZ PTH 893 45 3a' 3.2 0.2 1.43 f 9a
Data are expressed as mean f SEM for 7 to 10 rats per group. Statistically
significant differences, p<0.05. Baseline data
were not included in statistical analyses, and are shown for descriptive
purposes only.
a versus time-matched OVX
b versus time-matched hPTH (1-34)
versus time-matched sham
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TABLE VII
Bone Resorption Measures of the Right Proximal Tibia ir- Mature Ovariectomized
(OVX) Rats
Given Once Daily rec hPTH-(1-34) (LY333334), PTHrP-(1-36), or SDZ-PTH-893, a
PTHrP Analog,
for 30, 90, or 180. Days
Resorbing Surface Osteoclast Surface
E.PM Oc.PM
(%) (%)
Baseline OVX 20.3 1.4 9.0 0.8
Baseline Sham 18.3 1.8 6.1 1.0
30 Days
Sham 9.9 2.1 2.1 0.6
OVX 11.4 1.7 3.7 0.9
rhPTH(1-34) 8.6 1.2 2.3 0.6
PTHrP (1-36) 9.4 1.7 2.4 0.6
SDZ PTH 893 7,1 1,3a 1.6 0.6a
90 Days
Sham 7.0 1.0 1.6 0.5
OVX 9.6 2.0 4.5+1..1
rhPTH(1-34) 10.6 1.3 2.6 0.5
PTHrP (1-36) 10.3 1.5 3.1 0.6
SDZ PTH 893 10.0 1.3 2.0 0.7
180 Days
Sham 2.4 0.4 0.8 0.2
OYX 2.3 1.1 1.2 0.6
rhPTH(1-34) 6.4 1.5 2.0 0.4
PTHrP (1-36) 6.4 1.1a 2.0 0.6
SDZ PTH 893 3.0 1.0 0.4 0.3b
Data are expressed as mean SEM for 7 to 10 rats per group. Statistically
significant differences, p<0.05.
Baseline data were not included in statistical analyses, and are shown for
descriptive purposes only.
a versus time-matched OVX
b versus time-matched hPTH (1-34)
versus time-matched sham.
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TABLE VIII
Bone Mass ofthe Whole Body, Radius, or Femur in Mature Ovarlectomized Rats
Given Once Daily rec hPTH-(1-34) (LY333334),
PTHrP-(1-36), or SDZ PTH 893, a PTHrP Analog, for 30, 90, or 180 Days
Whole Body BMC Radius Ash Weight Left Femur
(mg) Length X Area BMC BMD
Baseline OVX' 66 1" 55 f 1 34.6 0.2 1.62 f 0.03 0.289 f 0.008 0.177
0.002
Baseline Sham2 53 f 1 52 t 1 33.6 10.2 1.55 t 0.03 0.294 f 0.007 0.190 0.004
30 Days
OVX 66 2* 58 2 34.9 0.3 1.60f0.05 0.289f0.014 0.180~=0.004
Sham 58 t 1 54 f 1 34.3 f 0.2 ' 1.57 f 0.03 0.300 f 0.009 0.190 f 0.004
rhPTH(1-34) 64 f 3 60 t 1' 34.9 f 0.3 1.68 0.04c 0.340 0.0118'c 0.200
0.004a ~
PTHrP(1-36) 67t2 58f1 34.7f0.2 1.64f0.03 0.3131: 0.011b 0.191f0.004 0
SDZ PTH 893 67 2 62 f 1 '' 34.9 f 0.3 1.73 f 0.048' 0.361 0.009''6 0.210
f 0.002a'b''
Ln
0
90 Days
OVX 68f4a 52f0 '35.5t0.4 1.65f0.02 0.293f0.006 0.178f0.003 ~
Sham 57f2 54f4 34.12= 0.3a 1.57f0.05 0.309f0.017 0.198f0.006a o
rhPTH(1-34) 69 f 3 66 f 1H' 35.7 f 0.2 1.73 f 0.03 0.401 f 0.010a' 0.232
f 0.005a' 0
PTHrP(1-36) 69 -L- 4 60 f 2a'c 35.5 0.2 1.71 f 0.04c 0.348 f 0.0078''
0.203 f 0.003"' o
SDZ PTH 893 70 f 2 69 f 1' 35.7 t 0.2 1.82 0.02~b, 0.442 0.006" 0.242
f 0.0033'
W
180 Days
OVX 72 4a 59f1 35.3 0.3 1.57f0.04 0.280f0.009 0.177f0:004
Sham 59 f 3 54 2 34.3 f 0.3a 1.53 0.04 0.291 0.009 0.189 0.004a
rhPTH(1-34) 71 f 3 72 f 23' 35.6 f 0.3' 1.84 f 0.04'' 0.451 f 0.0128' 0.234
f 0.004a"c
PTl-IrP(1-36) 68 2c 62 f 18'b=' 35.1 f 0.3c 1.64 f 0.036' 0.357 f 0.007"b
0.218 0.003a'=
SDZ PTH 893 68 f 3 78 :L3'' 36.4 f 0.3 1.96 f 0.05"' 0.530 0.018a"c 0.273
f 0.005"'
Abbreviations: X Area; BMC = bone mineral concentration; BMD = bone mineral
density; OVX = ovariectomized.
Data are expressed as mean f standard error of the mean (SEM) for 10 rats per
group. Statistically significant differences are shown in Table 3.
versus time-matched OVX
b versus time-matched PTH (1-34)
versus time-matched sham .
' OVX performed on day -1
2 BMC perfonned on day -30
Table IX
Gain in Body Weight and Serum Chemistries in Mature Ovariectomized Rats Given
Once Daily
rec hPTH-(1-34), PTHrP (1-36), or SDZ PTH 893, for 30, 90, or 180 Days
Body Serum Serum Serum Serum Serum Urea Kidney Alkaline
Weight Calcium Phosphate Magnesium Creatinine Nitrogen (mg Ca/gm Phosphatas
Gain (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) wet wt) e
(g) (IU/I.,)
Baseline --- 10.7=L0.1 7.7f0.3 2.91 0.07 0.74 0.01 16.5 0.6 0.20f0.01 78t5
Sham -- 10.6f0.1 7.3 0.3 2.75 0.08 0.76 0.01 18.2 0.6 0.25 0.04 100f4
Baseline OVX
30 Davs
Sham 1.0 f1.4 10.8f0.la 7.9f0.5 3.06f0.10 0.79f0.01 17.9f0.7 0.22f0.01 74f68
o
Ovx 1.7t3.0 10.4f0.1 7.3t0.2 2.94t0.08 0.78f0.02 17.7 0.9 0.27f0.03 88t3 Ln
PTH (1-34) 2.2f1.3 10.7f0.1a 6.5f0.2a 2.89f0.06 0.80f0.01 20.1t0.8a
0.25f0.02 83f3 N
PTHrP(1-36) 2.2f1.3 10.5t0.1 6.8t0.2 2.88t0.07 0.78f0.01 17.7f0.4 0.29f0.06
90f5' CD
~ SDZPTH893 -1.3f1.8 11.3f0.3abe 5.5f0.28b 290f0.07 0.79f0.01 19.1 0.6
0.26f0.01a 83f4
0
0
90 Days
0
Sham 13.1f3.8 10.2f0.1a 6.5f0.28 2.74f0.05 0.79f0.02 20.1f0.9 0.21f0.03 71f4a
Ovx 22.2f5.3 10.1f0.1 5.7f0.2 2.57t0.06 0.804:0.01 19.5t0.8 0.19t0.02 103t11 w
PTH (1-34) 30.6 f 4.8 10.7 f 0.180 5.4 f 0.20 2.70 0.03 0.7810.02 22.111.2
0.36 f 0.0680 1021 6'
PTHrP(1-36) 14.3 f 3.9 10.3 0.1b 5.4 t 0.3ab 2.60 0.08 0.78 f 0.02 21.3 f
1.3 0.21 0.03b 103 4
SDZPTH893 26.5f3.7 11.6t0.1ab 4.7t0.180 2.71f0.08 0.78t0.01 22.74:0.7a
0.35f0.0680 102 7c
180 Days
Sham 34.41: 10.4 10.6f0.18 7.10.4a 3.11f0.048 0.81f0.02 18.8t1.2 0.22f0.01 75
5 Ovx 52.3f13.0 10.2t0.2 6.2 0.3 2.75f0.08 0.81f0.03 18.2f0.9. 0.24f0.02 101 6
PTH(1-34) 41.4f16.2 10.9f0.28 6.0f0.3 2.80f0.070 0.75f0.02 19.8f0.8 0.24
0.028 108f50 PTHrP(1-36) 45.8f5.5 10.6f0.1a 6.010.2 2.6710.05 0.72f0.01a
17.4f0.8b 0.22f0.016 108f6
SDZ PTH 893 33.7 f 8.5 11.7 2= 0.6a 5.5 f 0.3ac' 2.90 f 0.08C 0.87 t 0.05b
20.0 f 1.0 1.28 0.60ab' 100 f 8'
Abbreviation: W W= Wet weight; OVX = ovariectomized.
Data are expressed as mean t standard error of the mean (SEM) for 10 rats per
group. Statistically siginificant differences, p<0.05.
a versus time-matched OVX
b versus time-matched hPTH (1-34)
versus time-matched sham
Table X
Biomechanical Measures of Strength of the Femur Neck and Mid-Diaphysis of
Mature OVX Rats Treated for 6 Months with hPTH 1-34 (PTH),
PTHrP-(1-36) (PTHrP), or the PTH Analog, SDZ PTH 893 '
Femur Neck Mid-Femur
Ultimate Cortical Moment Ultimate Stiffness Work to Ultimate Young's
Toiughness
Load, Thickness of Inertia Load Failure Stress Modulus
n (Fu) (t) m (Fu) .(S) (U) (Su) (E) (u)
(N) (mm) (mm4) (N) (N/mm) (mJ) (MPa) (Gpa)
J/m3
Baseline-Sham 10 113 6 0.64 0.01 4.2t0.2 156f4 460 16 56 4 204 5 7.9t0.3 -5.5
0.3
Baseline OVX 10 102t5 0.65t0.01 4.2t0.1 157 4 467 15 56 3 205 7 8.0 0.4
5.5t0.3
30 Davs
Sham 9 102t4 0.58 0.02 4.1f0.3 173 4 538t19 51 3 234 15 9.6 0.6 5.2t0.4
Ovx 10 114i-6 0.56 0.02 4.4 0.3 172t5 547 20 53t5 218t10 9.1t0.6 5.2t0.4
PTH 10 139 3a 0.64 0.02 80 4.5 0.2 185f6a 571 13 63t4 229t7 9.1t0.3 6.0 0.30
o
PTHrP 10 122 4e 0.58f0.01 b 4.6 0.1 182 5 576 17 61 3 223t6 8.9t0.3 5.8 0.3 v
SDZ 10 142t7 0.62 0.02 8 4.5 0.2 192t780 602 18a 68f5 237 68 9.5 0.3 6.5
0.4a' ~
CD
N N
90 Days
Sham 7 110 5 0.61t0.03 4.3 0.3 180 5 571f24a 49 3 23 i 8a 9.5 0.4 4.9 0.3 0
0 Ovx 9 117 5 0.52 0.03 4.6t0.3 164 6 484f22 46 4 198 8 7.5 0.3 4.4 0.4 0
PTH 10 146 4a 0.71 0.02a' 4.5F0.1 209 480 641t14$' 62 38 251t7a 10.0 0.2
5.7 0.3a w
PTHrP 10 137 38~ 0.64 0.01 a' 4.3t0.2 182f480 595 17a 51 2a 226 58C 9.8 0.3c
4.7 0.2a w
SDZ 10 157t580 0.78 0.02 k 4.6 0.2 221t4b 692 238b 65 3be 265t5' 10.6f0.3'
6.1f0.3b0
180 Davs
Sham 10 107t5 0.64 0.01 4.3 0.2 159 9 544 25 30f5 206t17 9.2 0.9 3.6 0.5
Ovx 6 113 4 0.61t0.02 4.5 0.2 158 10 563 26 38 4 188t11 8.8 0.4 3.5f0.4
PTH 9 147 6ac 0.89 0.01a' 5.4 0.380 241 9a 814f31ar 61t486 251 880 10.7 0.4a
5.0 0.3a' Croj
PTHrP 9 133 4abc 0.72 0.028b0 4.6 0.16 202 3ab' 767 73a' 53 4a 239 6a 11.9
1.280 4.7 0.4a
SDZ 6 165f8a0 1.02 0.02a0 6.2 0.680 256 13a 824 4880 69 68 250 13ao 9.6 0.6
5.7 0.5a0 Abbreviations: n number rats per group; OVX = ovariectomized
Data are shown as mean V SEM. Statistically significant differeces, p<0.05.
a versus time-matched OVX
b versus time-matched hPTH (1-34)
c versus time-matched sham
Table XI
Gain in Body Weight and Serum Chemistries in Mature,Ovariectomized Rats Given
Once Daily
rec hPTH-(1-34), PTHrP (1-36), or SDZ PTH 893, for 30, 90, or 180 Days
Body Serum Serum Serum Serum Serum Urea Kidney Alkaline
Weight Calcium Phosphate Magnesium Creatinine Nitrogen (mg Ca/gm Phosphatas
Gain (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) wet wt) e
(g) (IU/L)
Baseline -- 10.7 0.1 7.7f0.3 2.91 0.07 0.74 0.01 16.5 0.6 0.20 0.01 78 5
Sham --- 10.6 0.1 7.3 0.3 2.75+-0.08 0.76f0.01 18.2f0.6 0.25f0.04 100f4
Baseline OVX
30 Days
Sham 1.0 1.4 10.8t0.1 7.9t0.5 3.06f0.10 0.79f0.01 17.9f0.7 0.22t0.01 74f68 ~
0vx 1.7f3.0 10.4f0.1 7.3f0.2 2.94f0.08 0.78t0.02 17.7t0.9 0.27t0.03 88f3 0
PTH (1-34) 2.2 f 1.3 10.7 0.1a 6.5 f 0.280 2.89 0.06 0.80 f 0.01 20.1
0.8a 0.25 f 0.02 83 f 3 Ln
PTHrP(1-36) 2.2t1.3 10.5t0.1 6.8t0.2 2.88f0.07 0.78f0.01 17.7f0.4 0.29f0.06
90f5c o
SDZPTH893 -1.3f1.8 11.3f0.3abe 5.5f0.28b' 2.90t0.07 0.79f0.01 19.1t0.6
0.26t0.018 83 4
a~o
N
90 Days 0
0
Sham 13.1f3.8 10.2t0.18 6.5f0.28 2.74t0.05 0.79f0.02 20.1f0.9 0.21f0.03 71f4a
I
w
Ovx 22.2t5.3 10.1t0.1 5.7f0.2 2.57f0.06 0.80f0.01 19.5t0.8 0.19f0.02 103f11 0
PTH (1-34) 30.6f4.8 10.74. 0.1a 5.4f0.2 2.70f0.03 0.78f0.02 22.1f1.2
0.36f0.0680 102f60
iW
PTHrP(1-36) 14.3f3.9 10.3t0.1b 5.4f0.3ab 2.60f0.08 0.78t0.02 21.3t1.3
0.21t0.0313 103t4
SDZPTH893 26.5t3.7 11.6f0:1'b 4.7f0.180 2.71f0.08 0.78f0.01 22.7f0.7a
0.35f0.0680 102f70
180 Days
Sham 34.4f10.4 10.6t0.18 7.1t0.48 3.11f0.048 0.81t0.02 18.8f1.2 0.22f0.01
75:E5
Ovx 52.3f13.0 10.2 0.2 6.2f0.3 2.75f0.08 0..81f0.03 18.2f0.9 0.24f0.02 101 6
PTH(1-34) 41.4f16.2 10.9f0.2' 6.0f0.3c 2.80f0.07c 0.75f0.02 19.8f0.8
0.24f0.02a 1085
PTHrP(1-36) 45.8f5.5 10.6f0.1a 6.0f0.2 2.67f0.05c* 0.72f0.01ar 17.4f0.8b
0.22f0.01b 1086
SDZ PTH 893 33.7 f 8.5 11.7 f 0.6"c 5.5 f 0.3ar 2.90 0.08 0.87 f 0.05b 20.0
f 1.0 1.28 L 0.60ab 100 f 80 ~
Abbreviation: WW = Wet weight; OVX = ovariectomized.
Data are expressed as mean t standard error of the mean (SEM) for 10 rats per
group. Statistically siginificant differences, p<0.05.
' versus time-matched OVX
b versus time-matched hPTH (1-34)
versus time-matched sham
CA 02580281 2007-03-13
WO 2006/033912 PCT/US2005/032706
EQUIVALENTS
From the foregoing detailed description of the specific embodiments of the
invention, it
should be apparent that a unique method of administering PTHrP, or an analog
thereof, has been
described resulting in safe and efficacious treatment of osteoporosis that
minimizes the risk of, or
eliminates negative side effects, such as hypercalcemia *or the risk of
developing osteogenic
sarcomas. Although particular embodiments have been disclosed herein in
detail, this has been
done by way of example for purposes of illustration only, and is not intended
to be limiting with
respect to the scope of the appended claims that follow. In particular, it is
contemplated by the
inventor that substitutions, alterations, and modifications may be made to the
invention without
departing from the spirit and scope of the invention as defined by the claims.
For instance, the
choice of PTHrP analog, or the route of administration is believed to be
matter of routine for a
person of ordinary skill in the art with knowledge of the embodiments
described herein.
-84-
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
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