Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Modulators of Receptors for Parathyroid Hormone
and Parathyroid Hormone-Related Protein
Cross-Reference to Related Applications
This application is a continuation-in-part of U.S. Ser. No.
09/843,221, filed April 26, 2001, which claims the benefit of U.S.
Provisional Application No. 60/266,673, filed February 6, 2001, U.S.
Provisional Application No. 60/214,860, filed June 28, 2000,and U.S.
Provisional Application No. 60/200,053, filed April 27, 2000, which are
1o hereby incorporated by reference.
Field of the Invention
This invention relates to parathyroid hormone PTH), parathyroid
hormone-related protein (PTHrP) and modulators of PTH and PTHrP
receptors. This invention also relates to proteins modified for extended
half-life and, in particular, to proteins modified with polyethylene glycol.
Background of the Invention
PTH and PTHrP play important physiological roles in calcium
2o homeostasis and in development, respectively. Calcium concentration in
the blood is tightly regulated, due to the essential role of calcium in cell
metabolism. PTH is an endocrine hormone which is secreted from the
parathyroid gland in response to decreased serum calcium levels. PTH
acts directly to increase bone resorption and to stimulate renal calcium
reabsorption, thus increasing or preserving circulating calcium stores.
PTH also indirectly increases calcium absorption in the gut by stimulating
the renal hydroxylation of vitamin D.
Both primary and secondary hyperparathyroidism are conditions
that are associated with excessive levels of circulating parathyroid
3o hormone. Through the aforementioned pathways, excess PTH levels can
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cause hypercalcemia and osteopenia. Bone resorption inhibitors such as
bisphosphonates and OPG can effectively protect bone and can inhibit the
skeleton's contribution to hypercalcemia. However, the calcemic effects of
hyperparathyroidism on the kidney and gut are not addressed by
currently available therapy.
PTHrP is produced by many cell types, and plays an important role
in regulating skeletal development. Postnatally, the roles for PTHrP are
less clearly defined. Circulating levels of PTHrP are essentially non-
detectable in normal healthy adults. However, many tumors of diverse
to embryological origins produce and secrete PTHrP in quantities sufficient
to cause hypercalcemia. In fact, humoral hypercalcemia of malignancy
(HHM) is the most common paraneoplastic syndrome, which accounts for
significant patient morbidity and mortality.
Currently, HHM is treated with saline hydration followed by bone
resorption inhibitors such as bisphosphonates. This treatment regimen
typically takes 3-4 days to achieve significant reductions in serum calcium,
and the effects are relatively short-lived (less than one month). For patients
with high circulating levels of PTHrP, the effects of current treatment
options are even less impressive. Repeated administration of conventional
2o therapies are usually progressively less effective. These limitations to
current therapy strongly indicate an unmet medical need for rapid,
effective, and long-lasting treatments for HHM.
A major reason for the limited benefits of current HHM therapy is
the failure to directly inhibit PTHrP, which is very well established as the
principal pathophysiologic factor in HHM. Bone resorption inhibitors such
as bisphosphonates only inhibit bone resorption, while PTHrP also has
significant calcemic effects on the kidney and the gut. Total neutralization
of PTHrP would be the ideal adjuvant therapeutic approach to treatment
of HHM.
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Both PTH and PTHrP interact with PTH-1 receptor, which accounts
for most of their known effects. Mannstadt et al. (1999), Am. ]~. Ph, s
277. 5Pt 2. F665-75 (1999). Only PTH interacts with the newly discovered
PTH-2 receptor. Id. PTHrP can be changed to a PTH-2 receptor agonist,
however, by changing two residues to the residues at those positions in
PTH. Gardella et al. (1996), T. Biol. Chem. 271 (33):19888-93.
An N-terminal fragment of PTH has been used as a therapeutic
agent. Intermittently administered native PTH-(1-84) exhibits osteogenic
properties, and it has been recognized for decades that these properties
to can be fully realized with the C-terminally truncated fragment PTH-(1-34).
Both peptides bind and activate the PTH-1 receptor with similar affinities,
causing the activation of adenylate cyclase (AC) as well as phospholipase
C (PLC). AC activation through PTH-1 receptor generates cAMP, while
PLC activation through PTH-1 receptor generates PKC and intracellular
calcium transients. PTH-(1-34) can maximally activate both the AC and the
PLC pathways. It has been demonstrated that the anabolic effects of PTH-
(1-34) require short intermittent (daily) exposures Dobnig (1998),
Endocrinol.138: 4607-12. In human trials on postmenopausal women,
daily subcutaneous injection of low doses of PTH(1-34) were shown to
2o result in impressive bone formation in the spine and femoral neck with
significant reduction in incidence of vertebral fractures. These clinical data
reveal PTH as one of the most efficacious agents tested for osteoporosis.
Truncated PTH fragments have diminished AC/cAMP activation
and similarly diminished anabolic activity. Rixon et al. (1994), . Bone
Min. Res. 9:1179-89; Hilliker et al. (1996), Bone 19: 469-477; Lane et al.
(1996), T. Bone Min. Res. 11: 614-25. Such truncated PTH fragments have
this diminished activity(Rixon et al. (1994); Hilliker et al. (1996); Lane et
al.
(1996)) even if they maintain full agonism towards PKC. Rixon et al.,
(1994). These observations have led to the proposal that the AC/cAMP
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pathway is critical for the bone anabolic properties of PTH, while the
PLC/PKC pathway is dispensable in this regard. Rixon et al., (1994);
Whitfield et al. (1996), Calcified Tissue International 53: 81-7.
An opposing, but not mutually exclusive, theory suggests that PLC
activation (in addition to AC) might also be an important property of
anabolic PTH fragments. Takasu (1998), Endocrinol.139: 4293-9. The
apparent absence of PLC activation by some anabolic C-terminally
truncated PTH peptides may be an artifact of insensitive assay methods
combined with lower receptor binding. Takasu (1998). Progressive
1o truncations from the C-terminus of PTH-(1-34) result in stepwise
reductions in binding affinity for the PTH1R Takasu (1998). PKC
activation through PTH-1 receptor appears to be acutely sensitive to
binding affinity and to receptor density (Guo et al. (1995), Endocrinol 136:
3884-91), whereas cAMP activation is far less sensitive to these variables.
As such, hPTH-(1-31) has a slightly reduced (1-6 fold) affinity for PTH-1
receptor compared to hPTH-(1-34), while hPTH-(1-30) has a significantly
reduced (10-100 fold) affinity Takasu (1998). Perhaps due to this
decreased PTH-1 receptor affinity, PTH-(1-30) is a weak and incomplete
agonist for PLC activation via the rat PTH-1 receptor.
2o Compared to PTH-(1-34), PTH-(1-31) has similar or slightly reduced
anabolic potential (Rixon et al. (1994); Whitfield et al. (1996), Calcified
Tissue International 53: 81-7; Whitfield et al. (1996), Calcified Tissue
International 65: 143-7), binding affinity for PTH1R, and cAMP induction
(Takasu (1998)). PTH-(1-31) also has slightly reduced PLC activation.
Takasu (1998). In healthy humans, infusion of PTH-(1-31) and PTH-(1-34)
had similar stimulatory effects on plasma and urinary cAMP
concentration, but unlike PTH-(1-34), PTH-(1-31) failed to elevate serum
calcium, plasma 1,25(OH)2D3, or urinary N-TX levels. Fraher et al. (1999),
~. Clin. Endocrin. Met. 84: 2739-43. These data suggest that PTH-(1-31) has
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diminished capacity to induce bone resorption and to stimulation vitamin
D synthesis, which is a favorable profile for bone anabolic agents.
PTH-(1-30) was initially shown to lack anabolic properties
Whitfield et al. (1996), Calcified Tissue International 53: 81-7. More
recently, however, it has been demonstrated that PTH-(1-30) is anabolic
when administered at very high doses (400-2,000 ~ug/kg, vs. 80 ~g/kg for
PTH-(1-34)). The lower potency of PTH-(1-30) could be predicted by its
lower binding affinity fox PTH-1 receptor, its diminished cAMP activation,
and/or to its greatly diminished PKC activation. Takasu (1998). It remains
1o to be determined whether PTH-(1-30) has a similar or even more desirable
reduction in apparent bane resorption activity.
PTH-(1-28) is the smallest reported fragment to fully activate
CAMP. Neugebauer et al. (1995), Biochem. 34: 8835-42. However, hPTH-
(1-28) was initially reported to have no osteogenic effects in OVX rats.
Miller et al. (1997),J. Bone Min. Res. 12: S320 (Abstract). Recently, a very
high dose of PTH-(1-28) (1,000 ~g/kg/day) was shown to be anabolic in
OVX rats, whereas 200 ~.g/kg/day was ineffective. Whitfield et al. (2000),
T. Bone Min. Res. 15: 964-70. The diminished or absent anabolic effects of
some truncated PTH fragments has been attributed to rapid clearance in
2o vivo. Rixon et al. (1994).
Recombinant and modified proteins are an emerging class of
therapeutic agents. Useful modifications of protein therapeutic agents
include combination with the "Fc" domain of an antibody and linkage to
polymers such as polyethylene glycol (PEG) and dextran. Such
modifications are discussed in detail in a patent application entitled,
"Modified Peptides as Therapeutic Agents," U.S. Ser. No. 09/428,082, WO
00/24782, which is hereby incorporated by reference in its entirety.
A much different approach to development of therapeutic agents is
peptide library screening. The interaction of a protein ligand with its
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receptor often takes place at a relatively large interface. However, as
demonstrated for human growth hormone and its receptor, only a few key
residues at the interface contribute to most of the binding energy.
Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand
s merely displays the binding epitopes in the right topology or serves
functions unrelated to binding. Thus, molecules of only "peptide" length
(2 to 40 amino acids) can bind to the receptor protein of a given large
protein ligand. Such peptides may mimic the bioactivity of the large
protein ligand ("peptide agonists") or, through competitive binding,
to inhibit the bioactivity of the large protein ligand ("peptide
antagonists").
Phage display peptide libraries have emerged as a powerful
method in identifying such peptide agonists and antagonists. See, for
example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science
249: 404; U.S. Pat. No. 5,223,409, issued June 29,1993; U.S. Pat. No.
15 5,733,731, issued March 31,1998; U.S. Pat. No. 5,498,530, issued March 12,
1996; U.S. Pat. No. 5,432,018, issued July 11,1995; U.S. Pat. No. 5,338,665,
issued August 16,1994; U.S. Pat. No. 5,922,545, issued July 13,1999; WO
96/40987, published December 19,1996; and WO 98/15833, published
April 16,1998 (each of which is incorporated by reference in its entirety).
In such libraries, random peptide sequences are displayed by fusion with
coat proteins of filamentous phage. Typically, the displayed peptides are
affinity-eluted against an antibody-immobilized extracellular domain of a
receptor. The retained phages may be enriched by successive rounds of
affinity purification and repropagation. The best binding peptides may be
25 sequenced to identify key residues within one or more structurally related
families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in
which two distinct families were identified. The peptide sequences rnay
also suggest which residues may be safely replaced by alanine scanning or
by mutagenesis at the DNA level. Mutagenesis libraries may be created
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and screened to further optimize the sequence of the best binders.
Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.
Structural analysis of protein-protein interaction may also be used
to suggest peptides that mimic the binding activity of large protein
ligands. In such an analysis, the crystal structure may suggest the identity
and relative orientation of critical residues of the large protein ligand,
from which a peptide may be designed. See, e.g., Takasaki et al. (1997),
Nature Biotech. 15:1266-70. These analytical methods may also be used to
investigate the interaction between a receptor protein and peptides
to selected by phage display, which may suggest further modification of the
peptides to increase binding affinity.
Other methods compete with phage display in peptide research. A
peptide library can be fused to the carboxyl terminus of the lac repressor
and expressed in E. coli. Another E. coli-based method allows display on
z5 the cell's outer membrane by fusion with a peptidoglycan-associated
lipoprotein (PAL). Hereinafter, these and related methods are collectively
referred to as "E. coli display." In another method, translation of random
RNA is halted prior to ribosome release, resulting in a library of
polypeptides with their associated RNA still attached. Hereinafter, this
2o and related methods are collectively referred to as "ribosome display."
Other methods employ peptides linked to RNA; for example, PROfusion
technology, Phylos, Inc. See, for example, Roberts & Szostak (1997), Proc.
Natl. Acad. Sci. USA, 94:12297-303. Hereinafter, this and related methods
are collectively referred to as "RNA-peptide screening." Chemically
25 derived peptide libraries have been developed in which peptides are
immobilized on stable, non-biological materials, such as polyethylene rods
or solvent-permeable resins. Another chemically derived peptide library
uses photolithography to scan peptides immobilized on glass slides.
Hereinafter, these and related methods are collectively referred to as
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"chemical-peptide screening." Chemical-peptide screening may be
advantageous in that it allows use of D-amino acids and other unnatural
analogues, as well as non-peptide elements. Both biological and chemical
methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol.
3: 355-62. Conceptually, one may discover peptide mimetics of any
protein using phage display, RNA-peptide screening, and the other
methods mentioned above.
Summary of the Invention
1o The present invention concerns therapeutic agents that modulate
the activity of PTH and PTHrP. In accordance with the present invention,
modulators of PTH and PTHrP comprise:
a) a PTH/PTHrP modulating domain, preferably the amino
acid sequence of PTH/PTHrP modulating domains of PTH
15 and/or PTHrP, or sequences derived therefrom by phage
display, RNA-peptide screening, or the other techniques
mentioned above; and
b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc
domain, which is preferred;
2o wherein the vehicle is covalently attached to the carboxyl terminus of the
PTH/PTHrP modulating domain. The preferred vehicle is an Fc domain,
and the preferred Fc domain is an IgG Fe domain. Preferred PTH/PTHrP
modulating domains comprise the PTH and PTHrP-derived amino acid
sequences described hereinafter. Other PTH/PTHrP modulating domains
25 can be generated by phage display, RNA-peptide screening and the other
techniques mentioned herein. Such peptides typically will be antagonists
of both PTH and PTHrP, although such techniques can be used to generate
peptide sequences that serve as selective inhibitors (e.g., inhibitors of PTH
but not PTHrP).
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Further in accordance with the present invention is a process fox
making PTH and PTHrP modulators, which comprises:
a) selecting at least one peptide that binds to the PTH-1 or
PTH-2 receptor; and
s b) covalently linking said peptide to a vehicle.
The preferred vehicle is an Fc domain. Step (a) is preferably carried out by
selection from the peptide sequences in Tables 1A,1B, and 2 hereinafter or
from phage display, RNA-peptide screening, or the other techniques
mentioned herein.
to The compounds of this invention may be prepared by standard
synthetic methods, recombinant DNA techniques, or any other methods of
preparing peptides and fusion proteins. Compounds of this invention that
encompass non-peptide portions may be synthesized by standard organic
chemistry reactions, in addition to standard peptide chemistry reactions
zs when applicable.
The primary use contemplated for the compounds of this invention
is as therapeutic or prophylactic agents. The vehicle-linked peptide may
have activity comparable to-or even greater than-the natural ligand
mimicked by the peptide.
2o The compounds of this invention may be used for therapeutic or
prophylactic purposes by formulating them with appropriate
pharmaceutical carrier materials and administering an effective amount to
a patient, such as a human (or other mammal) in need thereof. Other
related aspects are also included in the instant invention.
2s Of particular interest in the present invention are molecules
comprising PTH/PTHRP modulating domains having a shortened PTH C-
terminal sequence, such as PTH-(1-28) or (1-34). The prior art shows no
anabolic studies using sustained duration delivery of such C-terminally
truncated PTH fragments. Although the art does not suggest it, molecules
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comprising smaller fragments such as PTH-(1-30)-Fc can be anabolic on
their own. Despite their weak agonism towards PLC (see Background of
the Invention), hPTH-(1-30) is nearly as effective at CAMP stimulation as is
hPTH-(1-34). While not wanting to be constrained by theory, the
inventors note that the anabolic properties of PTH fragments may be
selectively related to their cAMP activation, rather than PLC activation, so
that PTH fragments with reduced receptor affinity will have a favorable
anabolic profile. It is possible that continuous exposure to truncated PTH
fragments would have a different, and more favorable effect on bone
1o compared to continuous exposure to PTH-(1-34) or PTH-(1-84) that has
been demonstrated in humans by Fraher et al. (1999).
Numerous additional aspects and advantages of the present
invention will become apparent upon consideration of the figures and
detailed description of the invention.
Brief Description of the Figures
Figure 1 shows exemplary Fc dimers that may be derived from an
IgG1 antibody. "Fc" in the figure represents any of the Fc variants within
the meaning of "Fc domain" herein. "Xl" and "XZ" represent peptides or
linker-peptide combinations as defined hereinafter. The specific dimers
are as follows:
A: Single disulfide-bonded dimers. IgG1 antibodies typically have
two disulfide bonds at the hinge region between the constant and variable
domains. The Fc domain in Figure 1A may be formed by truncation
between the two disulfide bond sites or by substitution of a cysteinyl
residue with an unreactive residue (e.g., alanyl). In Figure 1A, the Fc
domain is linked at the C-terminus of the peptide.
B: Doubly disulfide-bonded dimers. This Fc domain may be formed
by truncation of the parent antibody to retain both cysteinyl residues in
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the Fc domain chains or by expression from a construct including a
sequence encoding such an Fc domain. In Figure 1B, the Fc domain is
linked at the C- terminus of the peptide.
C: Noncovalent dimers. This Fc domain may be formed by
elimination of the cysteinyl residues by either truncation or substitution.
One may desire to eliminate the cysteinyl residues to avoid impurities
formed by reaction of the cysteinyl residue with cysteinyl residues of other
proteins present in the host cell. The noncovalent bonding of the Fc
domains is sufficient to hold together the dimer.
to Other dimers may be formed by using Fc domains derived from different
types of antibodies (e.g., IgG2, IgM).
Figure 2 shows the structure of additional compounds of the
invention. Figure 2A shows a single chain molecule and may also
represent the DNA construct for the molecule. Figure 2B shows a dimer in
i5 which the linker-peptide portion is present on only one chain of the dimer.
Figure 2C shows a dimer having the peptide portion on both chains. The
dimer of Figure 2C will form spontaneously in certain host cells upon
expression of a DNA construct encoding the single chain as shown in
Figure 3. In other host cells, the cells could be placed in conditions
2o favoring formation of dimers or the dimers can be formed in vitro.
Figure 3 shows exemplary nucleic acid and amino acid sequences
(SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in
this invention.
Figure 4 shows the calcemic response of normal mice to PTH-(1-34)
25 and to PTH-(1-34)-Fe. Mice were challenged with vehicle (PBS, -X-), or
with PTH-(1-34) (open symbols) or with PTH-(1-34)-Fc (closed symbols).
Doses were 156 nmol/kg (circles), 469 nmol/kg (triangles) or 1,560
nmol/kg (squares). Data represent group means, n = 6 mice/group.
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Figure 5 shows that [AsnlO,Leul1]PTHrP-(7-34)-Fc inhibits the
calcemic response of normal mice to PTHrP. Normal male mice were
injected SC wifh vehicle (PBS, circles) or with human PTHrP-(1-34) at 0.5
mg/kg (squares). PTHrP-challenged mice were then immediately injected
SC with [AsnlO,LeuIIJPTHrP-(7-34)-Fc at 10 mg/kg (triangles) ox 30
mg/kg (diamonds). Data represent group means, with an n of 6
rnice/group.
Figure 6 shows the effect of [AsnlO,Leul1]PTHrP-(~-34)-Fc on
chronic hypercalcemia induced by PTH-(1-34)-Fc. Normal male mice were
to challenged once by SC injection with PTH-(1-34)-Fc (30 mg/kg) (open
circles), or with vehicle (PBS, open squares). Some PTH-(1-34)-Fc-
challenged mice were treated once, at the time of challenge, with
[AsnlO,Leul1]PTHrP-(7-34)-Fc at 10 (closed triangle), 30 (closed circle), or
100 mgjkg (closed square). All doses of [AsnlO,Leul1]PTHrP-(7-34)-Fc
15 caused a significant suppression of PTH-(1-34)-Fc-mediated
hypercalcemia. Data represent means ~ SEM, n = 5 mice/group.
Figure ~ shows CAMP accumulation in IZOS 1/2.8 rat osteoblast-
like cells. Cultures were treated with the phosphodiesterase inhibitor
IBMX and then challenged for 15 minutes with various PTH fragments.
2o CAMP was measured by ELISA.
Figure 8 shows the effects of single treatments on clinical chemistry.
Peripheral blood was obtained daily for 3 days following single
subcutaneous injections of the indicated compounds. Figure 8A shows
total serum calcium; Figure 8B, alkaline phosphatase (AP), a marker of
2~ osteoblast activity; Figure 8C, taxtrate-resistant acid phosphatase (TRAP),
a
marker of osteoclast activity, and Figure 8D, AP:TRAP ratio, an index of
relative osteoblas: osteoclast activity.
Figure 9 shows the effects of PTH constructs on bone mineral
density. Peripheral quantitative computed tomography (pQCT) was
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performed on the proximal tibial metaphysis of mice on day 15, after
injections of PTH constructs on day 0, 5 and 10.
Figure 10 shows the effect of twice-weekly PTH-(1-34)-Fc versus
daily PTH-(1-34) on tibial, trabecular, and cortical bone mineral density
(BMD). Daily PTH [PTH-(1-34)] was given at 80 ~g/kg/day (20
nmol/kg/day).
Figure 11 shows the effects of twice-weekly treatment on BMD and
serum calcium in aged ovariectomized (OVX) rats. Eleven months after
OVX, rats were treated twice per week with phosphate-buffered saline
1o (PBS, vehicle) or with APD (0.5 mg/kg) or with PTH-(1-34)-Fc (50
nmol/kg). DEXA was performed weekly. Blood was drawn 24 hours
after the second weekly injection, when the calcemic effects of PTH-Fe are
typically maximal.
Figure 12 shows the effect of a single subcutaneous injection of
15 PTH-(1-34)-Fc into OVX cynomologus monkeys. Monkeys were injected
with PTH-(1-34)-Fe at doses of 1-30 ~g/kg (n=1/group) or 100-1000 ~,g/kg
(n=2/group). Serum was analyzed for total calcium. The dotted line
indicates the threshold for hypercalcerrua, based on an elevation of
calcium greater than three standard deviations above the normal mean, on
20 two or more consecutive timepoints.
Figure 13 shows SDS-PAGE analysis of representative samples of
purified PEG-PTH (1-34) conjugate prepared from cysteine 27 PTH (1-34)
analog and 5 kD,10 kD, 20 kD and 30 kD linear PEG polymers, a 40 kD
branched polymer and the 8 kD bis-functional polymer respectively.
25 Figures 14A through 14D show cAMP response of murine MC3T3-
E1 osteoblast cultures to treatment with various PTH constructs. Cultures
were incubated with test materials for 10 minutes at room temperature.
Cell lysates were analyzed for cAMP production by ELISA.
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Figure 15 shows the hypercalcemic response of young male BDF1
mice to various PTH constructs. All treatments were single subcutaneous
injections of 300 nmol/kg (PTH referent).
Figure 16 shows the hypercalcemic response of young male BDF1
mice to a single subcutaneous injection of PTH-(1-34)-Fc or PEG-PTH
constructs. Blood ionized calcium was measured at the timepoints
indicated in the figure.
Figure 17 shows the effect of PTH-(1-34)-Fc or C27-30K PEG-PTH
on tibial bone mineral density (BMD) in adult male BDF1 mice. Mice were
to treated by subcutaneous. injection either once or twice per week for 4
weeks. The symbol # indicates significant difference from vehicle (PBS)-
treated mice, by two-way ANOVA.
Detailed Description of the Invention
15 Definition of Terms
The terms used throughout this specification are defined as follows,
unless otherwise limited in specific instances.
The term "comprising" means that a compound may include
additional amino acids on either or both of the N- or C- termini of the
2o given sequence. Of course, these additional amino acids should not
significantly interfere with the activity of the compound.
The term "acidic residue" refers to amino acid residues in D- or L-
form having sidechains comprising acidic groups. Exemplary acidic
residues include D and E.
25 The term "aromatic residue" refers to amino acid residues in D- or
L-form having sidechains comprising aromatic groups. Exemplary
aromatic residues include F, Y, and W.
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The term "basic residue" refers to amino acid residues in D- or L-
form having sidechains comprising basic groups. Exemplary basic
residues include H, K, and R.
The terms "hydrophilic residue" and "Haa" refer to amino acid
residues in D- or L-form having sidechains comprising at least one
hydrophilic functional group or polar group. Exemplary hydrophilic
residues include C, D, E, H, K, N, Q, R, S, and T.
The terms "lipophilic residue" and "Laa" refer to amino acid
residues in D- or L-form having sidechains comprising uncharged,
1o aliphatic or aromatic groups. Exemplary lipophilic sidechains include F, I,
L, M, V, W, and Y. Alanine (A) is amphiphilic-it is capable of acting as a
hydrophilic or lipophilic residue. Alanine, therefore, is included within the
definition of both "lipophilic residue" and "hydrophilic residue."
The term "nonfunctional residue" refers to amino acid residues in
z5 D- or L-form having sidechains that lack acidic, basic, or aromatic groups.
Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and
norleucine (Nle).
The term "vehicle" refers to a molecule that prevents degradation
and/or increases half-life, reduces toxicity, reduces immunogenicity, or
2o increases biological activity of a therapeutic protein. Exemplary vehicles
include an Fc domain (which is preferred) as well as a linear polymer (e.g.,
polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain
polymer (see, for example, U.S. Patent No. 4,289,872 to Denkenwalter et
al., issued September 15,1981; 5,229,490 to Tam, issued July 20,1993; WO
25 93/21259 by Freehet et al., published 28 October 1993); a lipid; a
cholesterol group (such as a steroid); a carbohydrate or oligosaccharide
(e.g., dextran); human serum albumin (HSA) and related molecules;
transtheratin (TTR) and related molecules; or any natural or synthetic
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protein, polypeptide or peptide that binds to a salvage receptor. Vehicles
are further described hereinafter.
The term "native Fc" refers to molecule or sequence comprising the
sequence of a non-antigen-binding fragment resulting from digestion of
whole antibody, whether in monomexic or multimeric form. The original
immunoglobulin source of the native Fc is preferably of human origin and
may be any of the immunoglobulins, although IgG1 and IgG2 are
preferred. Native Fc's are made up of monomeric polypeptides that may
be linked into dimeric or multimeric forms by covalent (i.e., disulfide
1o bonds) and non-covalent association. The number of intermolecular
disulfide bonds between monomeric subunits of native Fc molecules
xanges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgGl, TgG2, IgG3, IgAl, IgGA2). One example of a native Fc is a disulfide-
bonded dimer resulting from papain digestion of an IgG (see Ellison et al.
(1982), Nucleic Acids Res. 20: 4071-9). The term "native Fc' as used herein
is generic to the monomeric, dimeric, and multimeric forms.
The term "Fc variant" refers to a molecule or sequence that is
modified from a native Fc but still comprises a binding site for the salvage
receptor, FcRn. International applications WO 97/34631 (published 25
2o September 1997) and WO 96/32478 describe exemplary Fc variants, as
well as interaction with the salvage receptor, and are hereby incorporated
by reference in their entirety. Thus, the term "Fc variant" comprises a
molecule or sequence that is humanized from a non-human native Fc.
Furthermore, a native Fc comprises sites that may be removed because
they provide structural features or biological activity that are not required
for the fusion molecules of the present invention. Thus, the term "Fc
variant" comprises a molecule or sequence that lacks one or more native
Fc sites or residues that affect or are involved in (1) disulfide bond
formation, (2) incompatibility with a selected host cell (3) N-terminal
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heterogeneity upon expression in a selected host cell, (4) glycosylation, (5)
interaction with complement, (6) binding to an Fc receptor other than a
salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
Fc variants are described in further detail hereinafter.
The term "Fc domain' encompasses native Fc and Fc variant
molecules and sequences as defined above. As with Fc variants and native
Fc's, the term "Fc domain" includes molecules in monomeric or
multimeric form, whether digested from whole antibody or produced by
other means.
to The term "multimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two or more
polypeptide chains associated covalently, noncovalently, or by both
covalent and non-covalent interactions. IgG molecules typically form
dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers,
15 trimers, or tetramers. Multimers may be formed by exploiting the
sequence and resulting activity of the native Ig source of the Fc or by
derivatizing (as defined below) such a native Fc.
The term "dimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two polypeptide chains
2o associated covalently or non-covalently. Thus, exemplary dimers within
the scope of this invention are as shown in Figures 1 and 2.
The terms "derivatizing" and "derivative" or "derivatized"
comprise processes and resulting compounds respectively in which (1) the
compound has a cyclic portion; for example, cross-linking between
25 cysteinyl residues within the compound; (2) the compound is cross-linked
or has a cross-linking site; for example, the compound has a cysteinyl
residue and thus forms cross-linked dimers in culture or in vivo; (3) one or
more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-
terminus is replaced by -NRRI, NRC(O)Rl, -NRC(O)ORI, -NRS(O)zRl, -
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NHC(O)NHR, a succinimide group, or substituted or unsubstituted
benzyloxycarbonyl-NH-, wherein R and Rl and the ring substituents are
as defined hereinafter; (5) the C-terminus is replaced by -C(O)RZ or -NR3R4
wherein RZ, R3 and R4 are as defined hereinafter; and (6) compounds in
which individual amino acid moieties axe modified through treatment
with agents capable of reacting with selected side chains or terminal
residues. Derivatives are further described hereinafter.
The term "peptide" refers to molecules of 1 to ~5 amino acids, with
molecules of 5 to 34 amino acids preferred. Exemplary peptides may
1o comprise the PTH/PTHrP modulating domain of a naturally occurring
molecule or comprise randomized sequences.
The term "randomized" as used to refer to peptide sequences refers
to fully random sequences (e.g., selected by phage display methods or
RNA-peptide screening) and sequences in which one or more residues of a
is naturally occurring molecule is replaced by an amino acid residue not
appearing in that position in the naturally occurring molecule. Exemplary
methods for identifying peptide sequences include phage display, E. coli
display, ribosome display, RNA-peptide screening, chemical screening,
and the like.
2o The term "PTH/PTHrP modulating domain' refers to any amino
acid sequence that binds to the PTH-1 receptor and/or the PTH-2 receptor
and comprises naturally occurring sequences or randomized sequences.
Exemplary PTI-I/PTHrP modulating domains can be identified or derived
as described in the references listed for Tables 1A and 2, which are hereby
25 incorporated by reference in their entirety.
The term "PTH agonist" refers to a molecule that binds to PTH-1 or
PTH-2 receptor and increases or decreases one or more PTH activity assay
parameters as does full-length native human parathyroid hormone. An
exemplary PTH activity assay is disclosed in Example 1.
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The term "PTH antagonist" refers to a molecule that binds to PTH-1
or PTH-2 receptor and blocks or prevents the normal effect on those
parameters by full length native human parathyroid hormone. An
exemplary PTH activity assay is disclosed in Example 2.
The term "bone resorption inhibitor" refers to such molecules as
determined by the assays of Examples 4 and 11 of WO 97/23614:, which is
hereby incorporated by reference in its entirety. Exemplary bone
resorption inhibitors include OPG and OPG-L antibody, which are
described in WO 97/23614 and W098/46751, respectively, which are
1o hereby incorporated by reference in their entirety.
Additionally, physiologically acceptable salts of the compowlds of
this invention are also encompassed herein. By "physiologically
acceptable salts" is meant any salts that are known or later discovered to
be pharmaceutically acceptable. Some specific examples are: acetate;
15 trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;
sulfate; citrate; tartrate; glycolate; and oxalate.
Structure of compounds
In General.
Exemplary PTH and PTHrP receptor binding amino acid sequences
2o are described in Tables 1A,1B, and 2. Other information on PTH and
PTHxP is found in Mannstadt et al. (1999), Am.~. Ph_~siol. 277. 5Pt 2: F665-
75; and Gardella (1996), T. Biol. Chem. 271 (33): 19888-93. Each of these
references is hereby incorporated by reference in its entirety.
From the foregoing sequences, the present inventors identified in
25 particular preferred sequences derived from PTH and PTHrP. These
sequences can be randomized through the techniques mentioned above by
which one or mare amino acids may be changed while maintaining or
even improving the binding affinity of the peptide.
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In the compositions of matter prepared in accordance with this
invention, the peptide may be attached to the vehicle through the
peptide's C-terminus. Thus, the vehicle-peptide molecules of this
invention may be described by the following formula I:
I
P1-(Lx) -Fl
a
and multimers thereof, wherein:
Fl is a vehicle (preferably a PEG molecule) and is attached at the C-
terminus of Pl-(L1)a or through a sidechain at any residue from residue 14
1o through the C-terminal residue;
Plis a sequence of a PTH/PTHrP modulating domain;
Ll is a linker; and
ais0orl.
Peptides.
Any number of peptides may be used in conjunction with the
present invention. Peptides may comprise part of the sequence of
naturally occurring proteins, may be randomized sequences derived from
the sequence of the naturally occurring proteins, or may be wholly
randomized sequences. Phage display and RNA-peptide screening, in
2o particular, are useful in generating peptides for use in the present
invention.
A PTH/PTHrP modulating domain sequence particularly of
interest is of the formula
II
~5 ~N~8~10~11~12~~14~15~16~17~18~19~21~22~23~24~25~26~27~28~C
(SEQ ID NO: 3)
wherein:
XN is absent or is X3X4X5X6X', XzX3X4X5X6X', Xl7CzX3X4X5X6X', or
YX1X X3X ~5~6~~~
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X1 is an amino acid residue (nonfunctional, hydrophilic or aromatic
residue preferred; A, S or Y preferred);
XZ is an amino acid residue (nonfunctional residue preferred, V
most preferred);
X3 is an amino acid residue (hydrophilic residue preferred, S most
preferred);
X4 is an amino acid residue (acidic residue preferred, E most
preferred);
X5 is an amino acid residue (nonfunctional or basic residue
to preferred, H or I most preferred);
X6 is an amino acid residue (acidic or hydrophilic residue preferred,
Q or E most preferred);
X' is an amino acid residue (nonfunctional or aromatic residue
preferred, L or F most preferred);
15 X$ is an amino acid residue (nonfunctional residue preferred, M or
Nle most preferred);
Xl° is an amino acid residue (an acidic or hydrophilic residue
preferred, N or D most preferred);
X11 is an amino acid residue (nonfunctional or basic residue
2o preferred, L, R, or K most preferred);
X12 is an amino acid residue (nonfunctional or aromatic residue
preferred, G, F, or W most preferred);
X14 is an amino acid residue (basic or hydrophilic residue preferred,
H or S most preferred);
25 X15 is an amino acid residue (nonfunctional residue preferred, with
L or I most preferred);
X16 is an amino acid residue (nonfunctional or hydrophilic residue
preferred, Q, N, S, or A most preferred);
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X1' is an amino acid residue (acidic, hydrophilic, or nonfunctional
residue preferred; S, D, or L most preferred);
X1$ is an amino acid residue (nonfunctional residue preferred, M, L,
V or Nle most preferred);
X19 is an amino acid residue (acidic or basic residue preferred, E or
R most preferred);
X21 is an amino acid residue (nonfunctional residue or basic residue
preferred; V, M, R, or IVle most preferred);
X22 is an amino acid residue (hydrophilic, acidic, or aromatic
1o residue preferred, E or F most preferred);
X23 is an aromatic or lipophilic residue (W or P preferred);
X24 is a lipophilic residue (L preferred);
X25 is an amino acid residue (hydrophilic or basic residue preferred,
R or H most preferred);
is X26 is an amino acid residue (hydrophilic or basic residue preferred,
IC or H most preferred);
X2' is an amino acid residue (lipophilic, basic, or nonfunctional
residue preferred, K or L most preferred);
X2$ is an amino acid residue (lipophilic or nonfunctional residue
2o preferred, L or I most preferred);
X~ is absent ~r IS X29, X29X30, X29X30X31/ X29X30X31X32/ X29X30X31X32X33/
X29X30X31X32X33X34! X29X30X31X32X33X3AX35/ Or X29X30X31X32X33X34X35X36;
X29 is an amino acid residue (hydrophilic or nonfunctional residue
preferred, ~? or A most preferred);
25 X3° is an amino acid residue (hydrophilic or acidic residue
preferred, D or E most preferred);
X31 is an amino acid residue (lipophilic or nonfunctional residue
preferred, V or I most preferred);
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X3Z is an amino acid residue (basic residue preferred, H most
preferred);
X33 is an amino acid residue (hydrophilic residue preferred, N or T
most preferred);
S X34 is an amino acid residue (nonfunctional or aromatic residue
preferred, A, F or Y most preferred);
X35 is an amino acid residue (acidic residue preferred, E most
preferred);
X36 is an amino acid residue (aromatic residue preferred, Y most
io preferred) ;
provided that one or more of X14 through X36 is a cysteine residue.
A preferred PTH/PTHrP modulating domain sequence formula is
III
JNJ'JBHNLJ1ZICHLJ16SJ18J~9RJZ~EwLIUCKLJ~
z5 (SEQ ID NO: 4)
wherein:
jN is absent or is selected from jlJZJ3J4J5J6, JZJ3J4J5J6, J3J4J5J6;
Jl is an amino acid residue (nonfunctional, hydrophilic, or aromatic
residue preferred; A, S or Y most preferred);
2o J2 is an amino acid residue (nonfunctional residue preferred, V most
preferred);
J3 is an amino acid residue (hydrophilic residue preferred, S most
preferred);
J~ is an amino acid residue (acidic residue preferred, E most
25 preferred);
J5 is' an amino acid residue (nonfLUlctional residue preferred, I most
preferred);
J6 is an amino acid residue (basic residue preferred, Q preferred);
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J' is an amino acid residue (nonfunctional or aromatic residue
preferred, L or F most preferred);
J3 is an amino acid residue (nonfunctional residue preferred, M or
N1e most preferred);
J12 is an amino acid residue (nonfunctional or aromatic residue
preferred, G or W most preferred);
J16 is an amino acid residue (nonfunctional or hydrophilic residue
preferred, N, S, or A most preferred);
J13 is an amino acid residue (nonfunctional residue preferred, M,
to Nle, L, or V most preferred);
J19 is an acidic or basic residue (E or R preferred);
J21 is an amino acid residue (nonfunctional residue preferred, V, M,
or Nle most preferred);
J~ is absent or is J29/ J29J30/ J29J30J31/ J29J30J31J32/ J29J30J31J32J33/ ~r
J29J30J31J32J33J34;
J29 is an amino acid residue (hydrophilic or nonfunctional residue
preferred, Q or A most preferred);
J3~ is an amino acid residue (hydrophilic or acidic residue preferred,
D or E most preferred);
J31 is an amino acid residue (lipophilic or nonfunctional residue
2o preferred, V or I most preferred);
J32 is an amino acid residue (basic residue preferred, H most
preferred);
J33 is an amino acid residue (acidic residue preferred, N most
preferred);
J3ø is an amino acid residue (aromatic residue preferred, F or Y most
preferred);
provided that one or more of J14 through the C-terminal residue is a
cysteine residue.
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From the formula of SEQ ID NO: 4, peptides appearing in Table 1A
or 1B below are most preferred.
Another preferred PTH jPTHrP modulating domain sequence is
IV
ONLH01°011012ICSI015016LRRRFO~LHHLIO~
(SEQ ID NO: 5)
wherein:
ON is absent or is YO1OZO3O4O5O6O', OlOzO3O4O5O6O',
OZO3O4O5O6O', O3O4OSO6O', O4O5O6O', OSO6O , O6O', or O';
01 is an amino acid residue (nonfunctional residue preferred, A
most preferred);
OZ is an amino acid residue (nonfunctional residue preferred, V
most preferred);
03 is an amino acid residue (hydrophilic residue preferred, S most
preferred);
04 is an amino acid residue (acidic residue preferred, E most
preferred);
05 is an amino acid residue (basic or nonfunctional residue
preferred, H or I preferred);
06 is an amino acid residue (hydrophilic residue preferred, Q most
preferred);
O' is an amino acid residue (nonfunctional residue preferred, L
most preferred);
Ol° is an amino acid residue (acidic or hydrophilic residue
preferred, N or D most preferred);
011 is an amino acid residue (basic or nonfunctional residue
preferred, K or L most preferred);
Olz is an amino acid residue (aromatic or nonfunctional residue
preferred, G, F, or W most preferred);
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WO 2004/060386 PCT/US2002/036419
015 is an amino acid residue (hydrophilic or nonfunctional residue
preferred, I or S most preferred);
Olb is an amino acid residue (hydrophilic residue preferred, Q or N
most preferred);
Ol' is an amino acid residue (acidic or nonfunctional residue
preferred, D or L most preferred);
023 is an amino acid residue (aromatic residue preferred, with F or
W most preferred);
OC is absent ~r ZS 029, 029030/ 029030031/ 029030031032/ 029030031032033
029030031032033034 / ~29~30~31~32~33~34~35/ Or O29O30O31~32~33~34~35~36; and
029 through 036 are each independently amino acid residues;
provided that one or more of 014 through the C-terminal residue is a
cysteine residue.
From the formula of SEQ ID NO: 5, peptides appearing in Table 2
below are most preferred.
Exemplary peptide sequences for this invention appear in Tables
1A,1B and 2 below. These peptides may be prepared as described in the
cited references or in U.S. Pat. Nos. 4,423,037, 4,968,669, 5,001,~~., and
6,051,686, each of which is hereby incorporated by reference in its entirety,
or as described hereinafter. Molecules of this invention incorporating these
peptide sequences may be prepared by methods known in the art or as
described hereinafter. Single letter amino acid abbreviations are used.
Any of these peptides may be linked in tandem (i.e., sequentially), with or
without linkers. Any peptide containing a cysteinyl residue may be cross-
linked with another Cys-containing peptide, either or both of which may
be linked to a vehicle. Any peptide having more than one Cys residue may
form an intrapeptide disulfide bond, as well. Any of these peptides may
be derivatized as described hereinafter.
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Table 1A-PTH/PTHrP modulating domains based on PTH
Description Sequence SEQ
ID
N0:
human PTH(1-84)~ SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV10
ALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEA
DKADVNVLTKAKSQ
rat PTH(1-84)' AVSEIQLMHNLGKHLASVERMQWLRKKLQDVHNFV11
SLGVQMAAREGSYQRPTKKEDNVLVDGNSKSLGEG
DKADVDVLVKAKSQ
human PTH (7-84) LMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL12
APRDAGSQRPRKKEDNVLVESHEKSLGEADKADVN
VLTKAKSQ
human PTH(1-44) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV13
ALGAPLAPR
human PTH(1-38) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV14
ALG
human PTH(2-38) VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA15
LG
human PTH 1-34~' SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF16
AC 11 hUtTlatl PTH SVSEIQLMHNRGKHLNSMERVEWLRKKLQDVHNF17
1-34
L S11 hUP1'lafl PTH SVSEIQLMHNKGKHLNSMERVEWLRKKLQDVHNF18
1-34
AC 19 hUl'Tlan PTH SVSEIQLMHNLGKHLNSMRRVEWLRKKLQDVHNF19
1-34
T r1 human PTH 1-34 YVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF20
[Leu(8, 18), Tyr34~ SVSEIQLLHNLGKHLNSLERVEWLRKKLQDVHNY21
human
PTH 1-34
bovine PTH 1-34 AVSEIQFMHNLGKHLSSMERVEWLRKKLQDVHNF22
~L2U(8, 18), TYt'34] AVSEIQFLHNLGKHLSSLERVEWLRKKLQDVHNY23
bOVlnO PTH
1-34
orcine PTH 1-34 SVSEIQLMHNLGKHLSSLERVEWLRKKLQDVHNF24
ratPTH 1-34 AVSEIQLMHNLGKHLASVERMQWLRKKLQDVHNF25
[Leu (8, 21), Tyr34] AVSEIQLLHNLGKHLASVERLQWLRKKLQDVHNY26
rat PTH (1-
34 3
human PTH 1-31 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV27
Leu27 human PTH 1-31 SVSEIQLMHNLGKHLNSMERVEWLRKLLQDV28
Hendy et al. (1981), Proc. Natl. Acad. Sci USA 78: 7365; Kimura et al. (1983),
Biochem.
Bio_phys. Res. Commun.114: 493; Zanelli et al. (1985), EndocrinoloQV 117:
1962;
WW gender et al. (1985), J. Biol. Chem. 264: 4367.
Z Heinrich et al. (1984), J. Biol. Chem. 259: 3320.
3 Bachem Catalogue (1999).
A Doppelt et al. (1981), Calcif. Tissue Int. 33: 649; Podbesek et al. (1983)
Endocrinoloav 112:
1000; Kent et al. (1985), Clin. Sci. 68:171; McKee and Caulfield (1989),
Peptide Res. 2:161;
Lee and Russell (1989); Biopol, ers 28:1115; Reeve et al. (1990), Br. Med. T.
301: 314;
Neugebauer et al. (1994), Int. J. Peptide Protein Res. 43: 555.
Nakamura et al. (1981); Proc. Soc. Ex~. Biol. Med.168: 168; Law et al. (1983),
.T Clin.
Endocrinol. Metab. 56:1335; Wang et al. (1984), Eur. J. Pharmacol. 97,97, 209;
Sham et al.
(1986), Gen. Comp. Endocrinol. 61: 148; Smith et al. (1987), Arch. Biochem.
Bioph~253:
81.
~ Based on Coltrera et al. (1981), J. Biol. Chem. 256:10555; Bergeron et al.
(1981),
EndocrinoloQV 109: 1552.
Jouishomme et al. (1994), J. Bone Miner. Res. 9: 943; Whitfield and Morley;
TIPS 16: 382.
8 Barbier et al. (1997), T. Med. Chem. 40:1373.
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L2U 8,.18 ~ T r34 SEIQLLHNLGKHLNSLERVEWLRKKLQDVHNY29
PTH 3-34
bOVlne PTH 3-34 SEIQFMHNLGKHLSSMERVEWLRKKLQDVHNF30
~L2U(8, 18), Tyl'34] SEIQFLHNLGKHLSSLERVEWLRKKLQDVHNY31
bOVlne
PTH 3-34 1'
human PTH 7-34 LMHNLGKHLNSMERVEWLRKKLQDVHNF 32
~LeU(8, 18) Tyr34~hU~TlanLLHNLGKHLNSLERVEWLRKKLQDVHNY 33
PTH 7-34
bOVlne PTH 7-34 FMHNLGKHLSSMERVEWLRKKLQDVHNF 34
T r34 bovine PTH 7-34FMHNLGKHLSSMERVEWLRKKLQDVHNY 35
~LeU(8, 18), Tyr34] FLHNLGKHLSSLERVEWLRKKLQDVHNY 36
bOVlne
PTH 7-34 5'
[Leu(8, 18), Trpl2, FLHNLWKHLSSLERVEWLRKKLQDVHNY 37
Tyr34]
bovine PTH 7-34'6
[D-Trpl2, Tyr34] bOVlneFMHNL-D-Trp-KHLSSMERVEWLRKKLQDVHNY38
PTH 7-34 "
human PTH 1-30 SVSEIQLMHNLGKHLNSMERVEWLRKKLQD 39
AC 11 hUYTtan PTH SVSEIQLMHNRGKHLNSMERVEWLRKKLQD 40
1-30
L S11 hUlTlan PTH SVSEIQLMHNKGKHLNSMERVEWLRKKLQD 41
1-30
AC 19 hUITlan PTH SVSEIQLMHNLGKHLNSMRRVEWLRKKLQD 42
1-30
T r1 human PTH 1-30 YVSEIQLMHNLGKHLNSMERVEWLRKKLQD 43
LeU 8, 18 hUtTlan SVSEIQLLHNLGKHLNSLERVEWLRKKLQD 44
PTH 1-30
bOVlne PTH 1-3O AVSEIQFMHNLGKHLSSMERVEWLRKKLQD 45
L2U 8, 18 bOVlne PTH AVSEIQFLHNLGKHLSSLERVEWLRKKLQD 46
1-30
orcine PTH 1-30 SVSEIQLMHNLGKHLSSLERVEWLRKKLQD 47
1'at PTH 1-30 AVSEIQLMHNLGKHLASVERMQWLRKKLQD 48
[Leu(8, 21 ), Tyr34] AVSEIQLLHNLGKHLASVERLQWLRKKLQD 49
Pat
PTH 1-30
Leu27 human PTH 1-30 SVSEIQLMHNLGKHLNSMERVEWLRKLLQD 50
human PTH 1-29 SVSEIQLMHNLGKHLNSMERVEWLRKKLQ 51
human PTH 1-28 SVSEIQLMHNLGKHLNSMERVEWLRKKL 52
L2U 8, 18 PTH 3-3O SEIQLLHNLGKHLNSLERVEWLRKKLQD 53
bOVlne PTH 3-30 SEIQFMHNLGKHLSSMERVEWLRKKLQD 54
L8U 8, 18 bOVlne PTH SEIQFLHNLGKHLSSLERVEWLRKKLQD 55
3-30
human PTH 7-3O LMHNLGKHLNSMERVEWLRKKLQD 56
L2U 8, 18 hUnlan PTH LLHNLGKHLNSLERVEWLRKKLQD 57
7-30
bOVlne PTH 7-30 FMHNLGKHLSSMERVEWLRKKLQD 58
L2U 8, 18 bOVlne PTH FLHNLGKHLSSLERVEWLRKKLQD 59
7-30
~ Based on Schiparu et al. (1993), EndocrinoloQV 132: 2157-65.
1° Scharla et al. (1991), Horm. Metab. Res. 23: 66-9; McGowan et al.
(1983), Science 219: 67;
Lowik et al. (1985), Cell Calcium 6: 311.
" Based on Jobert et al. (1997), Endocrinolo~v 138: 5282; Schipani et al.
(1993); Rosenblatt
et al. (1977), J. Biol. Chem. 252: 5847; Segre et al. (1979), T_. Biol. Chem.
254: 6980;
Nussbaum et al. (1980), T. Biol. Chem. 225: 10183; Gray et al. (1980), Br. T.
Pharmac. 76:
259.
iz Nissenson et al. (1999), EndocrinoloQV 140: 1294-1300.
13 Jueppner et al. (1996), Endocrinolo~v.
is Horiuchi et al. (1983), Science 220: 1053.
is Schiparu et al. (1993); Holick et al. (1995), Bone 16:140S (abstract 223,
Conference,
Melbourne, February 1995).
ie Based on Dresner-Pollak et al. (1996), ~ 11:1061-5.
"Goldman et al. (1988), Endocrinolo~y 123: 2597.
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WO 2004/060386 PCT/US2002/036419
(Leu(8, 18), Trp12J FLHNLWKHLSSLERVEWLRKKLQD 60
bOVlne
PTH 7-30
D-Tr 12 k)OVlne PTH FMHNL-D-Trp-KHLSSMERVEWLRKKLQD 61
7-30
Table 1B-PTH/PTHrP modulating domains based on Cys
modifications of PTH
Description Sequence SEQ
ID
NO:
Cys33 PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWLR 172
C s-33 insertion KKLQD VHCNF
Cys27, 33 PTH(1-34)(Cys-27SVSEI QLMHN LGKHL NSMER VEWLR 173
re lacement, C s-33 KCLQD VHCNF
insertion
Cys-33 replacement SVSEI QLMHN LGKHL NSMER VEWLR 174
KKLQD VHCF
CGPTH 4 Cys-34 replacementSVSEI QLMHN LGKHL NSMER VEWLR 175
KKLQD VHNC
Cysl4 PTH(1-34) SVSEI QLMHN LGKCL NSMER VEWLR 176
KKLQD VHNF
Cysl5 PTH(1-34) SVSEI QLMHN LGKHC NSMER VEWLR 177
KKLQD VHNF
Cysl6 PTH(1-34) SVSEI QLMHN LGKHL CSMER VEWLR 178
KKLQD VHNF
Cys17 PTH(1-34) SVSEI QLMHN LGKHL NCMER VEWLR 179
KKLQD VHNF
Cysl8 PTH(1-34) SVSEI QLMHN LGKHL NSCER VEWLR 180
KKLQD VHNF
Cysl9 PTH(1-34) SVSEI QLMHN LGKHL NSMCR VEWLR 181
KKT~QD VHNF
Cys20PTH(1-34) SVSEI QLMININ LGKHL NSMEC VEWLR182
KKLQD VHNF
Cys21 PTH(1-34) SVSEI QLMHN LGKHL NSMER CEWLR 183
KKLQD VHNF
Cys22PTH(1-34) SVSEI QLM~IN LGKHL NSMER VCWLR184
KKLQD VHNF
Cys24PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWCR 185
KKLQD VHNF
Cys25PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWLC 186
KKT.QD VHNF
Cys26PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWLR 187
CKLQD VHNF
Cys27 PTH(1-34) SVSEI QLMHN LGItHL NSMER VEWLR188
KCLQD VHNF
Cys28PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWLR 189
KKCQD VHIVF
Cys29PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWLR 190
_K_KT_CD VHNF
Cys30PTH(1-34) SVSEI QLMHN LGKHL NSMER VEWLR 191
KKLQC VHNF
Cys31 PTH(1-34) SVSEI QLMHN LGKHL. NSMER VEWLR192
KKLQD CHNF
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Cys32PTH(1-34) ~ SVSEI QLMHIV LGKHf, NSMER VEWLR ~ 193
KKLOD V CNF
Table 2-PTH/PTHrP modulating domains based on PTHrP
Description Sequence SEQ
ID
NO:
human PTHPP(1-86)' AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAE62
IRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQ
ETNKVETYKEQPLKTP
human PTHrP 1-34 AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA63
[Tyr36] human PTHrP(1-36)AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAE64
Y
[IleS, Trp23, Tyr36] AVSEIQLLHDKGKSIQDLRRRFWLHHLIAEIHTAE65
hUITlan
PTHrP 1-36 3 Y
T r-human PTHPP 1-34 YAVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA66
[AsnlO, Leull, D-Phel2]AVSEHQLLHNL-D-Phe- 67
human PTHrP 1-34 ~9 KSIQDLRRRFFLHHLIAEIHTA
PTHrP 7-34 LLHDKGKSIQDLRRRFFLHHLIAEIHTA 68
[ASnlO, L2U11] hUnlanLLHNLGKSIQDLRRRFFLHHLIAEIHTA 69
PTHrP 7-34
Asnl6, Leul7 PTHrP LLHDKGKSINLLRRRFFLHHLIAEIHTA 70
7-34
[Leull, D-Trpl2] hUnlanLLHDL-D-Tr'p-KSIQDLRRRFFLHHLIAEIHTA71
PTHrP 7-34 22
[ASnlO, L0U11, D-Tr[712]LLHNL-D-Trp-KSIQDLRRRFFLHHLIAEIHTA72
PTHrP 7-34 ~3
D-Tr 12 PTHrP 8-34 LHNL-D-Trp-KSIQDLRRRFFLHHLIAEIHTA73
D-Phel2 PTHPP 8-34 LHNL-D-Phe-KSIQDLRRRFFLHHLIAEIHTA74
[ASnlO, L2U11, D-Trpl2]LLHNL-D-Trp-KSIQDLRRRFFLHHLIAEIHTA75
hUrTian
PTHrP 7-34 2
human PTHrP 1-30 AVSEHQLLHDKGKSIQDLRRRFFLHHLIAE 76
[Ile5, Trp23] hUrTlanAVSEIQLLHDKGKSIQDLRRRFWLHHLIAE 77
PTHrP 1-30
T r-human PTHfP 1-30 YAVSEHQLLHDKGKSIQDLRRRFFLHHLIAE7$
[AsnlO, Leull, D-Phel2]AVSEHQLLHNL-D-Phe- 79
human PTHrP 1-30 KSIQDLRRRFFLHHLIAE
PTHrP 7-30 LLHDKGKSIQDLRRRFFLHHLIAE 8O
[ASnlO, LAU11] hUri'1anLLHNLGKSIQDLRRRFFLHHLIAE $1
PTHrP 7-30
ASnl6, L0U17 PTHrP LLHDKGKSINLLRRRFFLHHLIAE $2
7-30
I8 Moseley et al. (1987), Proc. Natl. Acad. Sci. USA 84: 5048; Suva et al.
(1987), Science 237:
893; Kemp et al. (1987), Science 238: 1568; Paspaliaris et al. (1995), Bone
16: 141S (abstract
225, Conference, Melbourne 1995).
19 Based on JP 07316195, May 25,1994 (Nippon Kayaku).
zo Nagasaki et al. (1989), Biochem. Bioph~s. Res. Commun.158: 1036; Nutt et
al.;
EndocrinoloQV 127, 491 (1990).
zi Williams et al. (1998), T. Reproduction & Fertility 112: 59-67.
as Gardella et al. (1996), Endocrinol.137: 3936-41 ; Fukayama et al. (1998),
Am. T. Ph, s
274:E297-E303.
23 Li et al. (1996), Endocrinolo~v.
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(L2U11, D-Trpl2~ humanLLHDL-D-Trp-KSIQDLRRRFFLHHLIAE 83
PTHrP 7-30
(ASnlO, L2U11, D-Trpl2~LLHNL-D-Trp-KSIQDLRRRFFLHHLIAE 84
PTHrP 7-30
D-Tr 12 PTHI'P 8-30 LHNL-D-Trp-KSIQDLRRRFFLHHLIAE 85
D-Phel2 PTHrP 8-30 LHNL-D-Phe-KSIQDLRRRFFLHHLIAE 86
(ASnlO, L2U11, D-Trpl2~LLHNL-D-Trp-KSIQDLRRRFFLHHLIAE 87
human
PTHrP 7-30
[Haa(Laa Laa Haa Haa~2SVSEIQLMHNLGKHLNSMERVELLEKLLEKLHNF$$
Laa
22-31 human PTH 1-34
2a
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVELLEKLLKKLHNF$9
Laa
22-31 human PTH 1-34
2a
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVALAEALAEALHNF90
Laa
22-31 human PTH 1-34
25
[Haa(Laa Laa Haa Haa~2SVSEIQLMHNLGKHLNSMERVSLLSSLLSSLHNF91
Laa
22-31 human PTH 1-34
~s
[Haa(Laa Laa Haa Haa~2SVSEIQLMHNLGKHLNSMERVAFYDKVAEKLHNF92
Laa
22-31 human PTH 1-34
2'
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVELLEKLLEKLHNF 93
Laa
22-31 human PTH 7-34
24
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVELLEKLLKKLHNF 94
Laa
22-31 human PTH 7-34
2a
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVALAEALAEALHNF 95
Laa
22-31 human PTH 7-34
25
[Haa(Laa Laa Haa Haa~2LMHNLGKHLNSMERVSLLSSLLSSLHNF 96
Laa
22-31 human PTH 7-34
2s
[Haa(Laa Laa Haa Haa~2LMHNLGKHLNSMERVAFYDKVAEKLHNF 97
Laa
22-31 human PTH 7-34
2'
[Haa(Laa Laa Haa Haa~2AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA98
Laa
22-31 human PTHrP
1-34 24
[Haa(Laa Laa Haa Haa~2AVSEHQLLHDKGKSIQDLRRRELLEKLLKKLHTA99
Laa
22-31 human PTHrP
1-34 2
[Haa(Laa Laa Haa Haa~2AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA100
Laa
22-31 human PTHrP
1-34 2s
[Haa(Laa Laa Haa Haa~2AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA101
Laa
22-31 human PTHrP
1-34 2s
[Haa(Laa Laa Haa Haa~2AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA102
Laa
22-31 human PTHrP
1-34 2'
[Haa(Laa Laa Haa Haa~2LLHDKGKSIQDLRRRELLEKLLEKLHTA 103
Laa
22-31 human PTHrP
7-34 zs
[Haa(Laa Laa Haa Haa?2LLHDKGKSIQDLRRRELLEKLLKKLHTA 104
Laa
22-31 human PTHrP
7-34 24
[Haa(Laa Laa Haa Haa~2LLHDKGKSIQDLRRRALAEALAEALHTA 105
Laa
22-31 human PTHrP
7-34 z5
[Haa(Laa Laa Haa Haa~2LLHDKGKSIQDLRRRSLLSSLLSSLHTA 106
Laa
22-31 human PTHrP
7-34 2s
''~ Incorporating SEQ ID NO: 26 from U.S. Pat. No. 6,051,686.
zs ~corporating SEQ ID NO: 28 from U.S. Pat. No. 6,051,686.
z~ Incorporating SEQ ID NO: 29 from U.S. Pat. No. 6,051,686.
z~ Incorporating SEQ ID NO: 30 from U.S. Pat. No. 6,051,686.
z8 Incorporating SEQ ID NO: 26 from U.S. Pat. No. 6,051,686
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[Haa(Laa Laa Haa Haa)2LLHDKGKSIQDLRRRAFYDKVAEKLHTA 107
Laa
22-31 human PTHrP
7-34 z'
(LySll, LySl3; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA108
Arg2l;
Haa(Laa Laa Haa Haa)2
Laa
22-31 human PTHrP
1-34 zs
(Lysll, LySl3; Arg19,AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTS109
Arg2l;
Haa(Laa Laa Haa Haa)2
Laa
22-31 human PTHrP
1-34 30
(LySll, LYS13; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTAG110
Arg2l;
Haa(Laa Laa Haa Haa)2RR
Laa
22-31 human PTHrP
1-34 31
(LySll, LySl3; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLKEL111
Arg2l;
Haa(Laa Laa Haa Haa)2
Laa
22-31 human PTHrP
1-34 3z
[Lysll, Lysl3, A1a19,AVSEHQLLHDKGKSIQDLARRELLEKLLEKLHTA112
Arg2l,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 33
(LYS11, LySl3, Argl9,AVSEHQLLHDKGKS~QDLRRAELLEKLLEKLHTA113
A1a21,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 34
(LeUll, LySl3, Argl9,AVSEAQLLHDLGKSTQDLRRRELLEKLLEKLHAL114
Arg2l,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 3s
[Lysll, LySl3, Argl9,AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA115
Arg2l,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 3s
[Argll, Argl3, Arg19,AVSEHQLLHDRGRSIQDRRRELLERLLERLHTA116
Arg2l,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 3'
[Argll, LYS13, Argl9,AVSEHQLLHDRGKSIQDLRRRELLERLLKRLHTA117
Arg2l,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 3s
[Argll, Argl3, Argl9,AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA118
Arg2l,
Haa(Laa Laa Haa Haa)z
Laa
22-31 human PTHrP
1-34 3s
Haa(Laa Laa Haa Haa)zAVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA119
Laa
22-31 human PTHrP
1-34 40
Haa(Laa Laa Haa Haa)zAVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA120
Laa
22-31 human PTHrP
1-34 41
z~ Incorporating SEQ ID NO: 5 from U.S. Pat. No. 6,051,686.
so Based on SEQ ID NOS: 8, 9 from U.S. Pat. No. 6,051,686
3' Incorporating SEQ ID NO: 10 from U.S. Pat. No. 6,051,686
3z Ineorporating SEQ ID NO: 11 from U.S. Pat. No. 6,051,686
33 ~corporating SEQ ID NO: 12 from U.S. Pat. No. 6,051,686
3~ Incorporating SEQ ID NO: 12 from U.S. Pat. No. 6,051,686
ss Ineorporating SEQ ID N0:14 from U.S. Pat. No. 6,051,686
3G ~corporating SEQ ID NO: 15 from U.S. Pat. No. 6,051,686
3' Incorporating SEQ ID NO: 16 from U.S. Pat. No. 6,051,686
38 Incorporating SEQ ID N0:17 and 18 from U.S. Pat. No. 6,051,686
3~ Incorporating SEQ ID NO: 19 from U.S. Pat. No. 6,051,686
ao ~corporating SEQ ID NO: 20 from U.S. Pat. No. 6,051,686
ø1 Incorporating SEQ ID NO: 21 from U.S. Pat. No. 6,051,686
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Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA121
Laa
22-31 human PTHrP
1-34 42
Haa(Laa Laa Haa Haa)2AVSEIQFMHNLGKHLSSMERVELLEKLLEKLHNY122
Laa
22-31 human PTHrP
1-34 43
Haa(Laa Laa Haa Haa)2AVSEIQFMHNLGKHLSSMRRRELLEKLLEKLHNY123
Laa
22-31 human PTHrP
1-34 4a
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVELLEKLLEK 124
Laa
22-30 human PTH 1-30
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVELLEKLLKK 125
Laa
22-30 human PTH 1-30
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVALAEALAEA 126
Laa
22-30 human PTH 1-30
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVSLLSSLLSS 127
Laa
22-30 human PTH 1-30
[Haa(Laa Laa Haa Haa)2SVSEIQLMHNLGKHLNSMERVAFYDKVAEKLHNF128
Laa
22-30 human PTH 1-34
z'
[Haa(Laa Laa Haa Haa)zLMHNLGKHLNSMERVELLEKLLEK 129
Laa
22-30 human PTH 7-30
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVELLEKLLKK 130
Laa
22-30 human PTH 7-30
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVALAEALAEA 131
Laa
22-30 human PTH 7-30
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVSLLSSLLSS 132
Laa
22-30 human PTH 7-30
[Haa(Laa Laa Haa Haa)2LMHNLGKHLNSMERVAFYDKVAEK 133
Laa
22-30 human PTH 7-30
[Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRELLEKLLEK 134
Laa
22-30 human PTHrP
1-30
[Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRELLEKLLKK 135
Laa
22-30 human PTHrP
1-30
[Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRALAEALAEA 136
Laa
22-30 human PTHrP
1-30
[Haa(Laa Laa Haa Haa)~AVSEHQLLHDKGKSIQDLRRRSLLSSLLSS 137
Laa
22-30 human PTHrP
1-30
[Haa(Laa Laa Haa Haa)~AVSEHQLLHDKGKSIQDLRRRAFYDKVAEK 138
Laa
22-30 human PTHrP
1-30
[Haa(Laa Laa Haa Haa)2LLHDKGKSIQDLRRRELLEKLLEK 139
Laa
22-30 human PTHrP
7-30
[Haa(Laa Laa Haa Haa)2LLHDKGKSIQDLRRRELLEKLLKK 140
Laa
22-30 human PTHrP
7-30
[Haa(Laa Laa Haa Haa)~LLHDKGKSIQDLRRRALAEALAEA 141
Laa
22-30 human PTHrP
7-30
[Haa(Laa Laa Haa Haa)2LLHDKGKSIQDLRRRSLLSSLLSS 142
Laa
22-30 human PTHrP
7-30
[Haa(Laa Laa Haa Haa)zLLHDKGKSIQDLRRRAFYDKVAEK 143
Laa
22-30 human PTHrP
7-30
(LySll, LySl3; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLRK 144
Arg2l;
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
az ~eorporating SEQ ID NO: 22 from U.S. Pat. No. 6,051,686
ø3 Modified from SEQ ID NO: 23 from U.S. Pat. No. 6,051,686
4ø Modified from SEQ ID NO: 24 from U.S. Pat. No. 6,051,686
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~LySI l, LySl3; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLEK 145
Arg2l;
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
~LySI l, LYS13; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHT146
Arg2l;
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
~LySI l, LySl3; Argl9,AVSEHQLLHDKGKSIQDLRRRELLEKLLEK 147
Arg2l;
Haa(Laa Laa Haa Haa)z
Laa
22-30 human PTHrP
1-30
[Lysll, LySl3, A1a19,AVSEHQLLHDKGKSIQDLARRELLEKLLEK 148
Arg2l,
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
~LySl l, LySl3, Argl9,AVSEHQLLHDKGKSIQDLRRAELLEKLLEK 149
A1a21,
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
~L0u11, LYS13, Argl9,AVSEAQLLHDLGKSIQDLRRRELLEKLLEK 150
Arg2l,
Haa(Laa Laa Haa Haa)Z
Laa
22-30 human PTHrP
1-30
[Lysll, LySl3, Argl9,AVSEHQLLHDKGKSIQDLRRRELLERLLER 151
Arg2l,
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
[Argll, Argl3, Argl9,AVSEHQLLHDRGRSIQDRRRELLERLLER 152
Arg2l,
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
[Argil, Lysl3, Argl9,AVSEHQLLHDRGKSIQDLRRRELLERLLKR 153
ACg2l,
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
[Argil, Argl3, Argl9,AVSEHQLLHDRGRSIQDLRRRELLERLLKR 154
Arg2l,
Haa(Laa Laa Haa Haa)2
Laa
22-30 human PTHrP
1-30
Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRALAEALAEA 155
Laa
22-30 human PTHrP
1-30
Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRSLLSSLLSS 156
Laa
22-30 human PTHrP
1-30
Haa(Laa Laa Haa Haa)2AVSEHQLLHDKGKSIQDLRRRAFYDKVAEK 157
Laa
22-30 human PTHrP
1-30
Haa(Laa Laa Haa Haa)2AVSEIQFMHNLGKHLSSMERVELLEKLLEK 158
Laa
22-30 human PTHrP
1-30
Haa(Laa Laa Haa Haa)2AVSEIQFMHNLGKHLSSMRRRELLEKLLEK 159
Laa
22-30 human PTHrP
1-30
Another useful PTH/PTHrP modulating domain has the sequence
of the peptide known as TIP39:
SLALADDAAFRERARLLAALERRHWLNSYMHKLLVLDAP
(SEQ ID N0:160)
TIP39 is described by Usdin et al. (1999), Nature Neurosci. 2(11): 941-3;
Usdin et al. (1996), Endocrinolo~y 137(10): 4285-97; Usdin et al. (1995), j
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Biol. Chem. 270(26): 15455-8; Usdin et al. (1999), Endocrinol. 140(7): 3363-
71.
Additional useful PTH/PTHrP modulating domain sequences may
result from conservative and/or non-conservative modifications of the
amino acid sequences of SEQ ID NOS: 3, 4, 5, TIP39, or the sequences
listed in Tables 1A,1B, and 2. In particular, useful PTH/PTHrP
modulating domains furtller comprise molecules in which any residue at
position 14 through the C-terminal amino acid of any sequence in Tables
1A and 2 is substituted with a cysteine residue to provide an attachment
to site for a polymer (PEG preferred). Cysteine substitutions at position 27
through the C-terminal peptide are preferred.
Conservative modifications will produce peptides having
functional and chemical characteristics similar to those of the PTH or
PTHrP peptide from which such modifications are made. In contrast,
substantial modifications in the functional and/or chemical characteristics
of the peptides may be accomplished by selecting substitutions in the
amino acid sequence that differ significantly in their effect on maintaining
(a) the structure of the molecular backbone in the area of the substitution,
fox example, as a sheet or helical conformation, (b) the cllarge or
2o hydrophobicity of the molecule at the target site, or (c) the size of the
molecule.
For example, a "conservative amino acid substitution" may involve
a substitution of a native amino acid residue with a nonnative residue
such that there is little or no effect on the polarity or charge of the amino
acid residue at that position. Furthermore, any native residue in the
polypeptide may also be substituted with alanine, as has been previously
described for "alanine scanning mutagenesis" (see, for example,
MacLennan et al.,1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al.,
1998, Adv. Bioph~s. 35:1-24, which discuss alanine scanning mutagenesis).
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Desired amino acid substitutions (whether conservative or non-
conservative) can be determined by those skilled in the art at the time such
substitutions are desired. For example, amino acid substitutions can be
used to identify important residues of the peptide sequence, or to increase
or decrease the affinity of the peptide or vehicle-peptide molecules (see
preceding formulae) described herein. Exemplary amino acid
substitutions are set forth in Table 3.
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Table 3-Amino Acid Substitutions
Original Exemplary Preferred
Residues Substitutions Substitutions
Ala Val, Leu, lie Val
Arg Lys, Gln, Asn Lys
Asn Gln Gln
Asp Glu Glu
Cys Ser, Ala Ser
Gln Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gln, Lys, Arg
Arg
Ile Leu, Val, Met, Leu
Ala,
Phe, Norleucine
Leu Norleucine, Ile
Ife, Val,
Met, Ala, Phe
Lys Arg, 1,4 Diamino-Arg
butyric Acid,
Gln, Asn
Met Leu, Phe, Ile Leu
Phe Leu, Val, Ile, Leu
Ala, Tyr
Pro Ala Gly
Ser Thr, Ala, Cys Thr
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Phe
Ser
Val Ile, Met, Leu, Leu
Phe,
Ala, Norleucine
In certain embodiments, conservative amino acid substitutions also
encompass non naturally occurring amino acid residues which are
typically incorporated by chemical peptide synthesis rather than by
synthesis in biological systems.
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As noted in the foregoing section "Definition of Terms," naturally
occurring residues may be divided into classes based on common
sidechain properties that may be useful for modifications of sequence. For
example, non-conservative substitutions may involve the exchange of a
member of one of these classes for a member from another class. Such
substituted residues may be introduced into regions of the peptide that are
homologous with non-human orthologs, or into the non-homologous
regions of the molecule. In addition, one may also make modifications
using P or G for the purpose of influencing chain orientation.
to In making such modifications, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine
i5 (+1.8); glycine (-0.4); threonine (-0:7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine
(-
3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
The importance of the hydropathic amino acid index in conferring
interactive biological function on a protein is understood in the art. Kyte
2o et al., T. Mol. Biol.,157:105-131 (1982). It is known that certain amino
acids
may be substituted for other amino acids having a similar hydropathic
index or score and still retain a similar biological activity. In making
changes based upon the hydropathic index, the substitution of amino
acids whose hydropathic indices are within ~2 is preferred, those which
25 are within ~1 are particularly preferred, and those within ~0.5 are even
more particularly preferred.
It is also understood in the art that the substitution of like amino
acids can be made effectively on the basis of hydrophilicity. The greatest
local average hydrophilicity of a protein, as governed by the
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hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property of the
protein.
The following hydrophilicity values have been assigned to amino
acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ~ 1); glutamate
(+3.0 ~ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
threonine (-0.4); proline (-0.5 ~ 1); alanine (-0.5); histidine (-0.5);
cysteine (-
1.0); methionine (-1.3); valine (-1.5); leucine (-1.3); isoleucine (-1.3);
tyrosine
(-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based
1o upon similar hydrophilicity values, the substitution of amino acids whose
hydrophilicity values are within ~2 is preferred, those which are within ~1
are particularly preferred, and those within ~0.5 are even more
particularly preferred. One may also identify epitopes from primary
amino acid sequences on the basis of hydrophilicity. These regions are
also referred to as "epitopic core regions."
A skilled artisan will be able to determine suitable variants of the
polypeptide as set forth in the foregoing sequences using well known
techniques. For identifying suitable areas of the molecule that may be
changed without destroying activity, one skilled in the art may target
2o areas not believed to be important for activity. For example, when similar
polypeptides with similar activities from the same species or from other
species are known, one skilled in the art may compare the amino acid
sequence of a peptide to similar peptides. With such a comparison, one
can identify residues and portions of the molecules that are conserved
among similar polypeptides. It will be appreciated that changes in areas
of a peptide that are not conserved relative to such similar peptides would
be less likely to adversely affect the biological activity and/or structure of
the peptide. One skilled in the art would also know that, even in relatively
conserved regions, one may substitute chemically similar amino acids for
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the naturally occurring residues while retaining activity (conservative
amino acid residue substitutions). Therefore, even areas that may be
important for biological activity or for structure may be subject to
conservative amino acid substitutions without destroying the biological
activity or without adversely affecting the peptide structure.
Additionally, one skilled in the art can review structure-function
studies identifying residues in similar peptides that are important for
activity or structure. In view of such a comparison, one can predict the
importance of amino acid residues in a peptide that correspond to amino
to acid residues that are important for activity or structure in similar
peptides. One skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues of the
peptides.
One skilled in the art can also analyze the three-dimensional
structure and amino acid sequence in relation to that structure in similar
polypeptides. In view of that information, one skilled in the art may
predict the alignment of amino acid residues of a peptide with respect to
its three dimensional structure. One skilled in the art may choose not to
make radical changes to amino acid residues predicted to be on the surface
of the protein, since such residues may be involved in important
interactions with other molecules. Moreover, one skilled in the art may
generate test variants containing a single amino acid substitution at each
desired amino acid residue. The variants can then be screened using
activity assays know to those skilled in the art. Such data could be used to
gather information about suitable variants. For example, if one discovered
that a change to a particular amino acid residue resulted in destroyed,
undesirably reduced, or unsuitable activity, variants with such a change
would be avoided. In other words, based on information gathered from
such routine experiments, one skilled in the art can readily determine the
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amino acids where further substitutions should be avoided either alone or
in combination with other mutations.
A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):
422-427 (1996), Chou et al., Biochemistry,13(2): 222-245 (1974); Chou et al.,
Biochemistry,113(2): 211-222 (1974); Chou et al., Adv. Enzvmol. Relat.
Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:
251-276 and Chou et al., Bio h s. . 26: 367-384 (1979). Moreover,
computer programs are currently available to assist with predicting
to secondary structure. One method of predicting secondary structure is
based upon homology modeling. For example, two polypeptides or
proteins which have a sequence identity of greater than 30%, or similarity
greater than 40% often have similar structural topologies. The recent
growth of the protein structural data base (PDB) has provided enhanced
15 predictability of secondary structure, including the potential number of
folds within a polypeptide's or protein's structure. See Holm et al., Nucl.
Acid. Res., 27(1): 244-247 (1999). It has been suggested (Brenner et al.,
Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) that there are a limited number
of folds in a given polypeptide or_protein and that once a critical number
20 of structures have been resolved, structural prediction will gain
dramatically in accuracy.
Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997); Sippl
et al., Structure, 4(1): 15-9 (1996)), "profile analysis" (Bowie et al.,
Science,
25 253: 164-170 (1991); Gribskov et al., Meth. Enzym.,183: 146-159 (1990);
Gribskov et al., Proc. Nat. Acad, Sci., 84(13): 4355-8 (1987)), and
"evolutionary linkage" (See Home, supra, and Brenner s, upra).
Vehicles.
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This invention requires the presence of at least one vehicle (Fl)
attached to a peptide through the C-terminus or a sidechain of one of the
amino acid residues. Multiple vehicles may also be used; e.g., an Fc at the
C-terminus and a PEG group at a sidechain.
Fc domain. An Fc domain is the preferred vehicle. The Fc domain
may be fused to the C terminus of the peptides.
As noted above, Fe variants are suitable vehicles within the scope of
this invention. A native Fc may be extensively modified to form an Fc
variant in accordance with this invention, provided binding to the salvage
receptor is maintained; see, for example WO 97/34631 and WO 96/32478.
In such Fc variants, one may remove one or more sites of a native Fc that
provide structural features or functional activity not required by the fusion
molecules of this invention. One may remove these sites by, for example,
substituting or deleting residues, inserting residues into the site, or
truncating portions containing the site. The inserted or substituted
residues may also be altered amino acids, such as peptidomimetics or D-
amino acids. Fc variants may be desirable for a number of reasons, several
of which are described below. Exemplary Fc variants include molecules
and sequences in which:
1. Sites involved in disulfide bond formation are removed. Such removal
may avoid reaction with other cysteine-containing proteins present in
the host cell used to produce the molecules of the invention. For this
purpose, the cysteine-containing segment at the N-terminus may be
truncated or cysteine residues may be deleted or substituted with other
amino acids (e.g., alanyl, seryl). In particular, one may truncate the N
terminal 20-amino acid segment of SEQ ID NO: 2 or delete or
substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2.
Even when cysteine residues are removed, the single chain Fc domains
can still form a dimeric Fc domain that is held together non-covalently.
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2. A native Fc is modified to make it more compatible with a selected host
cell. Fox example, one may remove the PA sequence near the N-
terminus of a typical native Fc, which may be recognized by a digestive
enzyme in E. coli such as proline iminopeptidase. One may also add an
N-terminal methionine residue, especially when the molecule is
expressed recombinantly in a bacterial cell such as E. coli. The Fc
domain of SEQ ID NO: 2 is one such Fc variant.
3. A portion of the N-terminus of a native Fc is removed to prevent N-
terminal heterogeneity when expressed in a selected host cell. For this
1o purpose, one may delete any of the first 20 amino acid residues at the
N-terminus, particularly those at positions 1, 2, 3, 4 and 5.
4. One or more glycosylation sites are removed. Residues that are
typically glycosylated (e.g., asparagine) may confer cytolytic response.
Such residues may be deleted or substituted with unglycosylated
is residues (e.g., alanine).
5. Sites involved in interaction with complement, such as the C1q binding
site, are removed. For example, one may delete or substitute the EKK
sequence of human IgGl. Complement recruitment may not be
advantageous for the molecules of this invention and so may be
2o avoided with such an Fc variant.
6. Sites are removed that affect binding to Fc receptors other than a
salvage receptor. A native Fc may have sites for interaction with certain
white blood cells that are not required for the fusion molecules of the
present invention and so may be removed.
25 7. The ADCC site is removed. ADCC sites are known in the art; see, for
example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC
sites in IgGl. These sites, as well, are not required for the fusion
molecules of the present invention and so may be removed.
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8. When the native Fc is derived from a non-human antibody, the native
Fc may be humanized. Typically, to humanize a native Fc, one will
substitute selected residues in the non-human native Fc with residues
that are normally found in human native Fc. Techniques for antibody
humanization are well known in the art.
Preferred Fc variants include the following. In SEQ ID NO: 2
(Figure 4) the leucine at position 15 may be substituted with glutamate; the
glutamate at position 99, with alanine; and the lysines at positions 101 and
103, with alanines. In addition, one or more tyrosine residues can be
replaced by pllenyalanine residues.
An alternative vehicle would be a protein, polypeptide, peptide,
antibody, antibody fragment, or small molecule (e.g., a peptidomimetic
compound) capable of binding to a salvage receptor. For example, one
could use as a vehicle a polypeptide as described in U.S. Pat. No.
15 5,~39,2~~, issued April 14,1998 to Presta et al. Peptides could also be
selected by phage display or RNA-peptide screening for binding to the
FcRn salvage receptor. Such salvage receptor-binding compounds are also
included within the meaning of "vehicle" and are within the scope of this
invention. Such vehicles should be selected for increased half-life (e.g., by
2o avoiding sequences recognized by proteases) and decreased
immunogenicity (e.g., by favoring non-immunogenic sequences, as
discovered in antibody humanization).
Polymer vehicles. As noted above, polymer vehicles may also be
used for Fl and FZ. Various means for attaching chemical moieties useful as
25 vehicles are currently available, see, e.g., Patent Cooperation Treaty
("PCT") International Publication No. WO 96/11953, entitled "N-
Terminally Chemically Modified Protein Compositions and Methods,"
herein incorporated by reference in its entirety. This PCT publication
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discloses, among other things, the selective attachment of water soluble
polymers to the N-terminus of proteins.
A preferred polymer vehicle is polyethylene glycol (PEG). The
chemical modification of therapeutic proteins with polyethylene glycol
(PEG) has been broadly applied to improve the in vivo efficacy of protein
drugs. See "Protein Conjugates" chapter in Harris, et al., American
Chemical Society pp.118-216 (1997). PEGylation achieves this effect by
extending the drug's circulating half-life, increasing its solubility and in
some cases, reducing the drug's toxicity and immunogenicity.
1o The PEG group may be of any convenient molecular weight and
may be linear or branched. The average molecular weight of the PEG will
preferably range from about 2 kiloDalton ("kD") to about 100 kD. The
PEG group may also be attached to more than one therapeutic molecule;
for example, in a peptide-PEG-peptide configuration. Two PEG molecules
may also be attached to multiple sites or to a single site of a therapeutic
molecule; for example, two PEG molecules attached to a cysteine
sidechain through a linker. The average molecular weight of the PEG will
preferably range from about 2 kiloDalton ("kD") to about 100 kDa, more
preferably from about 5 kDa to about 50 kDa, most preferably from about
5 kDa, 20 kDa, or 30 kDa. In the present invention, linear monomethoxy
PEG-maleimides of molecular weights in the range of 5 - 30 kDa are
preferred, with 20-30 kDa polymers most preferred. Also preferred is a 40
kDa branched PEG-maleimide, comprised of two 20 kDa polymer "arms"
joined through a linker at the peptide attachment site. Another preferred
embodiment employs an 8 kDa bis-functional PEG-(maleimide)Z which can
be used to generate a peptide-PEG-peptide configuration.
A variety of conjugation chemistries have been investigated for
coupling PEG non-specifically to proteins. Zalipsky, Advanced Drug
Deliver~Reviews 16:157-182 (1995).. The PEG groups can generally be
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attached to the compounds of the invention via acylation or reductive
alkylation through a reactive group on the PEG moiety (e.g., an aldehyde,
amino, thiol, or ester group) to a reactive group on the inventive
compound (e.g., an aldehyde, amino, or ester group).
A useful strategy for the PEGylation of synthetic peptides consists
of combining, through farming a conjugate linkage in solution, a peptide
and a PEG moiety, each bearing a special functionality that is mutually
reactive toward the other. The peptides can be easily prepared with
conventional solid phase synthesis. The peptides are "preactivated" with
1o an appropriate functional group at a specific site. The precursors are
purified and fully characterized prior to reacting with the PEG moiety.
Ligation of the peptide with PEG usually takes place in aqueous phase and
can be easily monitored by reverse phase analytical HPLC. The PEGylated
peptides can be easily purified by preparative HPLC and characterized by
15 analytical HPLC, amino acid analysis and laser desorption mass
spectrometry.
Solid phase synthesis is also useful to prepare molecules PEGylated
through an available amino group (e.g., a lysine sidechain) using an
orthogonal protection strategy. In such synthesis, the peptide is
2o synthesized with removable protecting groups (e.g., Dde for lysine
sidechains) attached to the amino groups and any other reactive groups
that are not selected for PEGylation. The synthesized peptide can then
undergo a reaction resulting in PEGylation of the unprotected amino
group. The protecting groups on the other reactive groups can then be
25 removed by conventional means. This technique is especially useful for
PEGylation of one of the lysine residues of the PTH fragments mentioned
herein. For PTH(1-34), for example, the sidechain of one of the lysine
residues at positions 13, 26, or 27 is left unprotected while the others
comprise a Dde protecting group. The Dde groups are selectively removed
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using 2°l° hydrazine in water for 5 to 30 minutes at room
temperature. One
may use this technique to prepare a preferred molecule that is PEGylated
through the sidechain of the lysine residue at position 27.
Solid phase synthesis techniques may also be employed to prepare
molecules having a PEG moiety at the C-terminus. The molecule may
comprise a PEG moiety linking it to a resin used for solid phase synthesis.
The synthesized molecule may then be cleaved from the resin such that
the PEG moiety is retained with the peptide.
Site-directed approaches may be useful to maximize retention of
Zo biological activity while minimizing conjugate heterogeneity. Site-
directed PEGylation is typically achieved through a combination of
recombinant protein techniques and selective conjugation chemistries.
First, site-directed mutagenesis is used to introduce unique amino acids
with reactive ftmctional groups into the polypeptide sequence at positions
1s predicted to have minimal impact on protein activity Goodson, et al,
Bio/Technology 8:343-346 (1990) and Tsutsumi, et al., Proc. Natl. Acad.
Sci. 97:8548-8553 (2000). Cysteine is the preferred residue for engineering
directed conjugation sites because it is relatively scarce in proteins and the
thiol sidechain is among the most reactive of the protein nucleophiles.
2o Next, the mutagenic nucleic acid is introduced into a vector, which then is
used to transfect a host cell (e.g., E. coli, which is preferred), and the
peptide is expressed and isolated from the host cell. For the PEG portion
of the molecule, an activated monofunctional PEG polymer is prepared or
obtained commercially. Although there are a wide variety of activated
25 PEG polymers available which react specifically with cysteine thiols, such
as PEG-maleimide, -vinylsulfone, -iodoacetamide, -orthopyridyl-
disulphide and -epoxides, among others, the PEG-maleimides are by far
the most commonly used for conjugation. Zalipsky et al., pp. 347-370
(1992). Finally, the cysteine-containing protein and activated PEG are
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combined under appropriate reaction conditions to promote formation of
a PEG-protein conjugate which is subsequently purified and
characterized. PEG-maleimides are preferred PEGylating reagents, but
PEGylation may also be achieved with PEG-vinylsulfones, PEG-
S orthopyridyl-disulphides and PEG-iodoacetamides or any other activated
PEG that is selective for cysteine thiols.
Although cysteine residues are convenient substrates for
PEGylation chemistry, as noted above, the PTH-(1-34) peptide and other
peptides useful in this invention (e.g., see Tables 1A and 2) contains no
1o natural Cys residues for PEG conjugation. Cys mutations can be
introduced at any position, but structure-activity data suggests that C-
terminal domain insertions or substitutions are preferred. In one preferred
embodiment, cysteine residues were introduced into PTH-(1-34) by
substituting Cys for Lysine at position 27, and/or by inserting Cys
15 between histidine and asparagine at position 33.
Polysaccharide polymers are another type of water soluble polymer
which may be used for protein modification and may be prepared by
techniques generally as described above. Dextrans are polysaccharide
polymers comprised of individual subunits of glucose predominantly
20 linked by a1-6 linkages. The dextran itself is available in many molecular
weight ranges, and is readily available in molecular weights from about 1
kD to about 70 kD. Dextran is a suitable water soluble polymer for use in
the present invention as a vehicle by itself or in combination with another
vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The
2s use of dextran conjugated to therapeutic or diagnostic immunoglobulins
has been reported; see, for example, European Patent Publication No. 0
315 456, which is hereby incorporated by reference in its entirety. Dextran
of about 1 kD to about 20 kD is preferred when dextran is used as a
vehicle in accordance with the present invention.
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Linkers
Any "linker" group is optional. When present, its chemical
structure is not critical, since it serves primarily as a spacer. The linker
is
preferably made up of amino acids linked together by peptide bonds.
Thus, in preferred embodiments, the linker is made up of from 1 to 20
amino acids linked by peptide bonds, wherein the amino acids are selected
from the 20 naturally occurring amino acids. Some of these amino acids
may be glycosylated, as is well understood by those in the art. In a more
preferred embodiment, the 1 to 20 amino acids are selected from glycine,
to alanine, proline, asparagine, glutamine, and lysine. Even more preferably,
a linker is made up of a majority of amino acids that are sterically
unhindered, such as glycine and alanine. Thus, preferred linkers are
polyglycines (particularly (Gly)4, (Gly)5), poly(Gly-Ala), and polyalanines.
Other specific examples of linkers are:
is (Gly)3Lys(Gly)4 (SEQ ID NO: 6);
(Gly)3AsnGlySer(Gly)z (SEQ ID NO: 7);
(Gly)3Cys(Gly)4 (SEQ ID NO: ~); and
GlyProAsnGlyGly (SEQ ID NO: 9).
To explain the above nomenclature, for example, (Gly)3Lys(Gly)4 means
2o Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are also
preferred. The linkers shown here are exemplary; linkers within the scope
of this invention may be much longer and may include other residues.
Non-peptide linkers are also possible. For example, alkyl linkers
such as -NH-(CHZ)S C(O)-, wherein s = 2-20 could be used. These alkyl
25 linkers may further be substituted by any non-sterically hindering group
such as lower alkyl (e.g., C~ C6) lower acyl, halogen (e.g., Cl, Br), CN, NHZ,
phenyl, etc. An exemplary non-peptide linker is a PEG linker,
VI
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O
O O
'N O
H
wherein n is such that the linker has a molecular weight of 100 to 5000 kD,
preferably 100 to 500 kD. The peptide linkers may be altered to form
derivatives in the same manner as described above.
Derivatives
The inventors also contemplate derivatizing the peptide and/or
vehicle portion of tile compounds. Such derivatives may improve the
solubility, absorption, biological half life, and the like of the compounds.
1o The moieties may alternatively eliminate or attenuate any undesirable
side-effect of the compounds and the like. Exemplary derivatives include
compounds in which:
1. The compound or some portion thereof is cyclic. For example, the
peptide portion may be modified to contain two or more Cys residues
15 (e.g., in the linker), which could cyclize by disulfide bond formation.
2. The compound is cross-linked or is rendered capable of cross-linking
between molecules. For example, the peptide portion may be modified
to contain one Cys residue and thereby be able to form an
intermolecular disulfide bond with a like molecule. The compound
2o may also be cross-linked through its C-terminus, as in the molecule
shown below.
V
O
F1-~X1 )b-CC3-N NH
2
F1- X1 -CO-N v NH
( )b
O
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3. One or more peptidyl [-C(O)NR-] linkages (bonds) is replaced by a
non-peptidyl linkage. Exemplary non-peptidyl linkages are -CHZ
carbamate [-CH2 OC(O)NR-], phosphonate , -CHz sulfonamide [-CHz
S(O)ZNR-], urea [-NHC(O)NH-], -CHz secondary amine, and alkylated
peptide [-C(O)NR6- wherein R6 is lower alkyl].
4. The N-terminus is derivatized. Typically, the N-terminus may be
acylated or modified to a substituted amine. Exemplary N-terminal
derivative groups include -NRRI (other than -NHz), -NRC(O)R1,
-NRC(O)OR1, -NRS(O)ZRI, -NHC(O)NHRI, succinimide, or
1o benzyloxycarbonyl-NH- (CBZ-NH-), wherein R and Rl are each
independently hydrogen or lower alkyl and wherein the phenyl ring
may be substituted with 1 to 3 substituents selected from the group
consisting of C~ C4 alkyl, C~ C4 alkoxy, chloro, and bromo.
5. The free C-terminus is derivatized. Typically, the C-terminus is
15 esterified or amidated. Exemplary C-terminal derivative groups
include, for example, -C(O)R2 wherein R2 is lower alkoxy or -NR3R~
wherein R3 and R4 are independently hydrogen or C~ C$ alkyl
(preferably Ci C4 alkyl).
6. A disulfide bond is replaced with another, preferably more stable,
2o cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al.
(1996), I. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am.
Pep. Sip., 357-9.
7. One or more individual amino acid residues is modified. Various
derivatizing agents are known to react specifically with selected
25 sidechains or terminal residues, as described in detail below.
Lysinyl residues and amino terminal residues may be reacted with
succinic or other carboxylic acid anhydrides, which reverse the charge of the
lysinyl residues. Other suitable reagents for derivatizing alpha-amino-
containing residues include imidoesters such as methyl picolinimidate;
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pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic
acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction
with glyoxylate.
Arginyl residues may be modified by reaction with any one or
combination of several conventional reagents, including phenylglyoxal, 2,3-
butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl
residues requires that the reaction be performed in alkaline conditions
because
of the high pICa of the guanidine functional group. Furthermore, these
reagents
may react with the groups of lysine as well as the arginine epsilon-amino
1o group.
Specific modification of tyrosyl residues has been studied extensively,
with particular interest in introducing spectral labels into tyrosyl residues
by
reaction with aromatic diazonium compounds or tetranitromethane. Most
commonly, N-aeetylimidizole and tetranitromethane are used to form O-acetyl
tyrosyl species and 3-vitro derivatives, respectively.
Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively
modified by reaction with carbodiimides (R'-N=C=N-R') such as 1-cyclohexyl-
3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-
dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues
2o may be converted to asparaginyl and glutaminyl residues by reaction with
ammonium ions.
Glutaminyl and asparaginyl residues may be deamidated to the
corresponding glutamyl and aspartyl residues. Alternatively, these residues
are deamidated under mildly acidic conditions. Either form of these residues
falls within the scope of this invention.
Cysteinyl residues can be replaced by amino acid residues or other
moieties either to eliminate disulfide bonding or, conversely, to stabilize
cross-
linking. See, e.g., Bhatnagar et al. (1996), T. Med. Chem. 39: 3814-9.
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Derivatization with bifunctional agents is useful for cross-linking the
peptides or their functional derivatives to a water-insoluble support matrix
or
to other macromolecular vehicles. Commonly used cross-linking agents
include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-
hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid,
homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-
maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-
azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are
1o capable of forming crosslinks in the presence of light. Alternatively,
reactive
water-insoluble matrices such as cyanogen bromide-activated carbohydrates
and the reactive substrates described in LT.S. Pat. Nos. 3,969,287; 3,691,016;
4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein
immobilization.
z5 Carbohydrate (oligosaccharide) groups may conveniently be
attached to sites that are known to be glycosylation sites in proteins.
Generally, O-linked oligosaccharides are attached to serine (Ser) or
threonine (Thr) residues while N-linked oligosaccharides are attached to
asparagine (Asn) residues when they are part of the sequence Asn-X-
2o Ser/Thr, where X can be any amino acid except proline. X is preferably
one of the 19 naturally occurring amino acids other than proline. The
structures of N-linked and O-linked oligosaccharides and the sugar
residues found in each type are different. One type of sugar that is
commonly found on both is N-acetylneuraminic acid (referred to as sialic
25 acid). Sialic acid is usually the terminal residue of both N-linked and O-
linked oligosaccharides and, by virtue of its negative charge, may confer
acidic properties to the glycosylated compound. Such sites) may be
incorporated in the linker of the compounds of this invention and are
preferably glycosylated by a cell during recombinant production of the
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polypeptide compounds (e.g., in mammalian cells such as CHO, BHK,
COS). However, such sites may further be glycosylated by synthetic or
semi-synthetic procedures known in the art.
Other possible modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of Beryl or threonyl residues,
oxidation of the sulfur atom in Cys, methylation of the alpha-amino
groups of lysine, arginine, and histidine side chains. Creighton, Proteins:
Structure and Molecule Properties (W. H. Freeman & Co., San Francisco),
pp. 79-86 (1983).
1o Compounds of the present invention rnay be changed at the DNA
level, as well. The DNA sequence of any portion of the compound may be
changed to codons more compatible with the chosen host cell. For E. coli,
which is the preferred host cell, optimized codons are known in the art.
Codons may be substituted to eliminate restriction sites or to include silent
restriction sites, which may aid in processing of the DNA in the selected
host cell. The vehicle, linker and peptide DNA sequences may be modified
to include any of the foregoing sequence changes.
Methods of Making
The compounds of this invention largely may be made in
2o transformed host cells using recombinant DNA techniques. To do so, a
recombinant DNA molecule coding for the peptide is prepared. Methods
of preparing such DNA molecules are well known in the art. For instance,
sequences coding for the peptides could be excised from DNA using
suitable restriction enzymes. Alternatively, the DNA molecule could be
synthesized using chemical synthesis techniques, such as the
phosphoramidate method. Also, a combination of these techniques could
be used.
The invention also includes a vector capable of expressing the
peptides in an appropriate host. The vector comprises the DNA molecule
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that codes for the peptides operatively linked to appropriate expression
control sequences. Methods of effecting this operative linking, either
before or after the DNA molecule is inserted into the vector, are well
known. Expression control sequences include promoters, activators,
enhancers, operators, ribosomal binding sites, start signals, stop signals,
cap signals, polyadenylation signals, and other signals involved with the
control of transcription or translation.
The resulting vector having the DNA molecule thereon is used to
transform an appropriate host. This transformation may be performed
1o using methods well known in the art.
Any of a large number of available and well-known host cells may
be used in the practice of this invention. The selection of a particular host
is dependent upon a number of factors recognized by the art. These
include, for example, compatibility with the chosen expression vector,
toxicity of the peptides encoded by the DNA molecule, rate of
transformation, ease of recovery of the peptides, expression characteristics,
bio-safety and costs. A balance of these factors must be struck with the
understanding that not all hosts may be equally effective for the
expression of a particular DNA sequence. Within these general guidelines,
2o useful microbial hosts include bacteria (sueh as E. coli sp.), yeast (such
as
Saccharomyces sp.) and other fungi, insects, plants, mammalian (including
human) cells in culture, or other hosts known in the art.
Next, the transformed host is eultured and purified. Host cells may
be cultured under conventional fermentation conditions so that the
desired compounds are expressed. Such fermentation conditions are well
known in the art. Finally, the peptides are purified from culture by
methods well known in the art.
The compounds may also be made by synthetic methods. For
example, solid phase synthesis techniques may be used. Suitable
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techniques are well known in the art, and include those described in
Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and
Panayotis eds.); Merrifield (1963), T. Am. Chem. Soc. 85: 2149; Davis et al.
(1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase
Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins
(3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2:
257-527. Solid phase synthesis is the preferred technique of making
individual peptides since it is the most cost-effective method of making
small peptides.
1o Compounds that contain derivatized peptides or which contain
non-peptide groups may be synthesized by well-known organic chemistry
techniques.
Uses of the Compounds
The compounds of this invention have pharmacologic activity
resulting from their interaction with PTH-1 receptor or PTH-2 receptor.
Mannstadt et al. (1999), Am. T. Ph~~. 277. 5Pt 2. F665-75. PTH and
agonists thereof increase bone resorption, increase renal calcium
reabsorption, decrease epidermal proliferation, and decrease hair growth.
2o Holick et al. (1994) Proc. Natl. Sci. USA 91 (17): 8014-6; Schilli et al.
(1997),
Invest. Dermatol.108(6): 928-32. Thus, antagonists of PTH-1 receptor
and/or PTH-2 receptor are useful in treating:
~ primary and secondary hyperparathyroidism;
~ hypercalcemia, including hypercalcemia resulting from solid
tumors (breast, lung and kidney) and hematologic malignacies
(multiple myeloma, lymphoma and leukemia); idiopathic
hypercalcernia, and hypercalcemia associated with
hyperthyroidism and renal function disorders;
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~ tumor metastases, particularly metastases to bone, and particularly
related to breast and prostate cancer;
~ cachexia and anorexia, particularly as associated with cancer;
~ osteopenia that is related to or aggravated by aberrant PTH
receptor signaling, including various forms of osteoporosis, such as:
- primary osteoporosis;
- post-menopausal and age-related osteoporosis;
- endocrine osteoporosis (hyperthyroidism, hyperparathyroidism,
Cushing's syndrome, and acromegaly);
- hereditary and congenital forms of osteoporosis (e.g.,
osteogenesis imperfecta, homocystinuria, Menkes' syndrome,
and Riley-Day syndrome);
- osteoporosis due to immobilization of extremities;
- osteoporosis secondary to other disorders, such as
z5 hemochromatosis, hyperprolactinemia, anorexia nervosa,
thyrotoxicosis, diabetes mellitus, celiac disease, inflammatory
bowel disease, primary biliary cirrhosis, rheumatoid arthritis,
ankylosing spondylitis, multiple myeloma, lymphoproliferative
diseases, and systemic mastocytosis;
- osteoporosis secondary to surgery (e.g., gastrectomy) or to drug
therapy, such as chemotherapy, anticonvulsant therapy,
immunosuppressive therapy, and anticoagulant therapy;
- osteoporosis secondary to glucocorticosteroid treatment for such
diseases as rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE), asthma, temporal arteritis, vasculitis,
chronic obstructive pulmonary disease, polymyalgia
rheumatica, polymyositis, chronic interstitial lung disease;
- osteoporosis secondary to glucocorticosteroid and/or
immunomodulatory treatment to prevent organ rejection
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following organ transplant such as kidney, liver, lung, heart
transplants;
- osteoporosis due to submission to microgravity, such as
observed during space travel;
- osteoporosis associated with malignant disease, such as breast
cancer, prostate cancer;
~ Paget's disease of bone (osteitis deformans) in adults and juveniles;
~ osteomyelitis, or an infectious lesion in bone, leading to bone loss;
~ osteopenia following surgery, induced by steroid administration,
1o and associated with disorders of the small and large intestine and
with chronic hepatic and renal diseases.
~ Osteonecrosis, or bone Bell death, associated with traumatic injury
or nontraumatic necrosis associated with Gaucher's disease, sickle
cell anemia, systemic lupus erythematosus, rheumatoid arthritis,
periodontal disease, osteolytic metastasis, and other conditions;
~ alopecia (deficient hair growth or partial or complete hair loss),
including androgenic alopecia (male pattern baldness), toxic
alopecia, alopecia senilis, alopecia areata, alopecia pelada, and
trichotillomania;
2o and the like.
There are other conditions wherein a patient would benefit from the
activity of PTH or PTHrP. For those indications, PTH receptor agonists are
useful as a therapeutic treatment. In particular, such indications include
fracture repair (including healing of non-union fractures), osteopenia,
including various forms of osteoporosis, such as:
- primary osteoporosis;
- post-menopausal and age-related osteoporosis;
- endocrine osteoporosis (hyperthyroidism, Cushing's syndrome,
and acromegaly);
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- hereditary and congenital forms of osteoporosis (e.g.,
osteogenesis imperfecta, homocystinuria, Menkes' syndrome,
and Riley-Day syndrome);
- osteoporosis due to immobilization of extremities;
- osteoporosis secondary to other disorders, such as
hemochromatosis, hyperprolactinemia, anorexia nervosa,
thyrotoxicosis, diabetes mellitus, celiac disease, inflammatory
bowel disease, primary biliary cirrhosis, rheumatoid arthritis,
ankylosing spondylitis, multiple myeloma, lymphoproliferative
Zo diseases, and systemic mastocytosis;
- osteoporosis secondary to surgery (e.g., gastrectomy) or to drug
therapy, such as chemotherapy, anticonvulsant therapy,
immunosuppressive therapy, and anticoagulant therapy;
- osteoporosis secondary to glucocorticosteroid treatment for
15 diseases such as RA, SLE, asthma, temporal arteritis, vasculitis,
chronic obstructive pulmonary disease, polymyalgia
rheumatica, polymyositis, chronic interstitial lung disease;
- osteoporosis secondary to glucocorticosteroid and/or
immunomodulatory treatment to prevent organ rejection
2o following organ transplant such as kidney, liver, lung, heart
transplants;
- osteoporosis due to submission to microgravity, such as
observed during space travel;
- osteoporosis associated with malignant disease, such as breast
25 cancer, prostate cancer;
PTH agonists with extended half-life (e, g., those linked to Fc domains)
may be used with an inhibitor of bone resorption. Inhibitors of bone
resorption include OPG and OPG derivatives, OPG-L (RANICL) antibody,
calcitonin (e.g., Miacalcin~, Calcimar~), bisphosphonates (e.g., APD,
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alendronate, risedronate, etidronate, pamidronate, tiludronate, clodronate,
neridronate, ibandronate, zoledronate), estrogens (e.g., Premarin~,
Estraderm~, Prempro~, Alora~, Climara0, Vivelle~, Estratab~ Ogen~),
selective estrogen receptor modulators (e.g., raloxifene, droloxifene,
lasofoxifene), tibolone, and the like. Exemplary bone resorption inhibitors
are described in W093/46751 and W097/23614, which are hereby
incorporated by reference in their entirety.
The compounds of this invention may be appropriate as a
monotherapy for the treatment of Osteoporosis, and it is possible that the
1o addition of an antiresorptive agent to PTH-Fc treatment will increase both
their efficacy and therapeutic window. Both PTH and PTH-Fc cause an
increase in both bone formation and bone resorption. The ability of
antiresorptives to block the osteoclast response could limit the
hypercalcemic effects of PTH-Fc and could also increase bone mas
Pharmaceutical Compositions
In General. The present invention also provides methods of using
pharmaceutical compositions of the inventive compounds. Such
pharmaceutical compositions may be for administration for injection, or for
oral, pulmonary, nasal, transdermal or other forms of administration. In
2o general, the invention encompasses pharmaceutical compositions comprising
effective amounts of a compound of the invention together with
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers,
adjuvants and f or carriers. Such compositions include diluents of various
buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;
additives such as detergents and solubilizing agents (e.g., Tween ~0,
Polysorbate ~0), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g.,
lactose, mannitol); incorporation of the material into particulate
preparations of
polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into
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liposomes. Hyaluronic acid may also be used, and this may have the effect of
promoting sustained duration in the circulation. Such compositions may
influence the physical state, stability, rate of in vivo release, and rate of
in vivo
clearance of the present proteins and derivatives. See, e.g., Remin~;ton's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA
18042) pages 1435-1712 which are herein incorporated by reference in their
entirety. The compositions may be prepared in liquid form, or may be in dried
powder, such as lyophilized form. Implantable sustained release formulations
are also contemplated, as are transdermal formulations.
1o Twice weekly dosing of the compounds of this invention is superior to
daily injection of PTH (1-34) for increasing osteoblast number, bone volume,
and bone mineral density in rodents. In adult mice, twice weekly dosing with
PTH-(1-34)-Fc caused greater increases in bone density and bone volume
compared to daily PTH-(1-34). (See Figure 10.) In an aged OVX rat model of
i5 osteoporosis, twice weekly dosing was able to reverse more than 50% of the
bone loss induced by one year of estrogen ablation. The effect seen in the
aged
rat model was even greater when combined with a bisphosphonate (APD). In
rats, a single SC injection of PTH-(1-34)-Fc (340 nmol/kg) caused a
hypercalcemic response which persisted for 72 hours (Figure 8). This duration
2o is concordant with the rate of clearance of PTH-(1-34)-Fc from the serum,
and is
consistent with an optimal twice-weekly dosing regimen in rats.
The optimal dosing of primates may be less frequent compared to rats or
mice. Weekly (or less frequent) dosing may be optimal in primates, based on
the observation that the hypercalcemic response of OVX eynomolgus monkeys
25 to a single subcutaneous injection of PTH-(1-34)-Fc (10-34 nmol/kg)
persisted
for about 168 hours (Figure 11). This observation suggests that a single
subcutaneous dose of PTH-(1-34)-Fc in primates is cleared within about 1 week,
which could also represent the maximum dosing frequency required for
anabolic effects.
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Oral dosage forms. Contemplated for use herein are oral solid
dosage forms, which are described generally in Chapter 89 of Remington's
Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton PA
18042, which is herein incorporated by reference in its entirety. Solid
dosage forms include tablets, capsules, pills, troches or lozenges, cachets
or pellets. Also, liposomal or proteinoid encapsulation may be used to
formulate the present compositions (as, for example, proteinoid
microspheres reported in U.S. Patent No. 4,925,673). Liposomal
encapsulation may be used and the liposomes may be derivatized with
to various polymers (e.g., U.S. Patent No. 5,013,556). A description of
possible solid dosage forms for the therapeutic is given in Chapter 10 of
Marshall, IC., Modern Pharmaceutics (1979), edited by G. S. Banker and C.
T. Rhodes, herein incorporated by reference in its entirety. In general, the
formulation will include the inventive compound, and inert ingredients
which allow for protection against the stomach environment, and release
of the biologically active material in the intestine.
Also specifically contemplated are oral dosage forms of the above
inventive compounds. If necessary, the compounds may be chemically
modified so that oral delivery is efficacious. Generally, the chemical
2o modification contemplated is the attachment of at least one moiety to the
compound molecule itself, where said moiety permits (a) inhibition of
proteolysis; and (b) uptake into the blood stream from the stomach or
intestine. Also desired is the increase in overall stability of the compound
and increase in circulation time in the body. Moieties useful as covalently
attached vehicles in this invention may also be used for this purpose.
Examples of such moieties include: PEG, copolymers of ethylene glycol
and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and
Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981),
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Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, , pp. 367-
83; Newmark, et al. (1982), T-Appl. Biochem. 4:185-9. Other polymers that
could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred
for pharmaceutical usage, as indicated above, axe PEG moieties.
Fox oral delivery dosage forms, it is also possible to use a salt of a
modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]
amino) caprylate (SNAC), as a carrier to enhance absorption of the
therapeutic compounds of this invention. The clinical efficacy of a heparin
formulation using SNAC has been demonstrated in a Phase II trial
1o conducted by Emisphere Technologies. See US Patent No. 5,792,451, "Oral
drug delivery composition and methods".
The compounds of this invention can be included in the
formulation as fine multiparticulates in the form of granules or pellets of
particle size about 1 mm. The formulation of the material for capsule
~5 administration could also be as a powder, lightly compressed plugs or
even as tablets. The therapeutic could be prepared by compression.
Colorants and flavoring agents may all be included. For example,
the protein (or derivative) may be formulated (such as by liposome or
microsphere encapsulation) and then further contained within an edible
2o product, such as a refrigerated beverage containing colorants and
flavoring agents.
One may dilute or increase the volume of the compound of the
invention with an inert material. These diluents could include
carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose,
25 sucrose, modified dextrans and starch. Certain inorganic salts may also be
used as fillers including calcium triphosphate, magnesium carbonate and
sodium chloride. Some commercially available diluents are Fast-Flo,
Emdex, STA-Rx 1500, Emcompress and Avicell.
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Disintegrants may be included in the formulation of the therapeutic
into a solid dosage form. Materials used as disintegrants include but are
not limited to starch including the commercial disintegrant based on
starch, Explotab. Sodium starch glycolate, Amberlite, sodium
carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange
peel, acid carboxymethyl cellulose, natural sponge and bentonite may all
be used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
1o tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
Binders may be used to hold the therapeutic agent together to form
a hard tablet and include materials from natural products such as acacia,
tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl
15 cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone
(PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
An antifrictional agent may be included in the formulation of the
therapeutic to prevent sticking during the formulation process. Lubricants
2o may be used as a layer between the therapeutic and the die wall, and these
can include but are not limited to; stearic acid including its magnesium
and calcium salts, polytetrafluoroetllylene (PTFE), liquid paraffin,
vegetable oils and waxes. Soluble lubricants may also be used such as
sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of
25 various molecular weights, Carbowax 4000 and 6000.
Glidants that might improve the flow properties of the drug during
formulation and to aid rearrangement during compression might be
added. The glidants may include starch, talc, pyrogenic silica and
hydrated silicoaluminate.
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To aid dissolution of the compound of this invention into the
aqueous environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl sulfate,
dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic
detergents might be used and could include benzalkonium chloride or
benzethonium chloride. The list of potential nonionic detergents that
could be included in the formulation as surfactants are lauromacrogol 400,
polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and
60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid
ester, methyl cellulose and carboxymethyl cellulose. These surfactants
could be present in the formulation of the protein or derivative either
alone or as a mixture in different ratios.
Additives may also be included in the formulation to enhance
uptake of the compound. Additives potentially having this property are
15 for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
Controlled release formulation may be desirable. The compound of
this invention could be incorporated into an inert matrix which permits
release by either diffusion or leaching mechanisms e.g., gums. Slowly
degenerating matrices may also be incorporated into the formulation, e.g.,
2o alginates, polysaccharides. Another form of a controlled release of the
compounds of this invention is by a method based on the Oros therapeutic
system (Alza Corp.), i.e., the drug is enclosed in a semipermeable
membrane which allows water to enter and push drug out through a
single small opening due to osmotic effects. Some enteric coatings also
25 have a delayed release effect.
Other coatings may be used for the formulation. These include a
variety of sugars which could be applied in a coating pan. The therapeutic
agent could also be given in a film coated tablet and the materials used in
this instance are divided into 2 groups. The first are the nonenteric
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materials and include methyl cellulose, ethyl cellulose, hydroxyethyl
cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols. The second group consists of the
enteric materials that are commonly esters of phthalic acid.
A mix of materials might be used to provide the optimum film
coating. Film coating may be carried out in a pan coater or in a fluidized
bed or by compression coating.
Pulmonary delivery forms. Also contemplated herein is pulmonary
to delivery of the present protein (or derivatives thereof). The protein (or
derivative) is delivered to the lungs of a mammal while inhaling and
traverses across the lung epithelial lining to the blood stream. (Other
reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et
al.
(1990), Internatl. j. Pharmaceutics 63:135-44 (leuprolide acetate); Braquet
15 et al. (1989), T. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146
(endothelin-
1); Hubbard et al. (1989), Aruzals Int. Med. 3: 206-12 (a1-antitrypsin); Smith
et al. (1989), T. Clin. Invest. 84: 1145-6 (od-proteinase); Oswein et al.
(March
1990), "Aerosolization of Proteins", Proc. Sip. Resp. Drug Deliver~I,
Keystone, Colorado (recombinant human growth hormone); Debs et al.
20 (1988), ~. Immunol. 140: 3482-8 (interferon-y and tumor necrosis factor a)
and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating
factor).
Contemplated for use in the practice of this invention axe a wide
range of mechanical devices designed for pulmonary delivery of
25 therapeutic products, including but not limited to nebulizers, metered
dose inhalers, and powder inhalers, all of which are familiar to those
skilled in the art. Some specific examples of commercially available
devices suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the
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Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colorado; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler
powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.
All such devices require the use of formulations suitable for the
dispensing of the inventive compound. Typically, each formulation is
specific to the type of device employed and may involve the use of an
appropriate propellant material, in addition to diluents, adjuvants
andjor carriers useful in therapy.
1o The inventive compound should most advantageously be
prepared in particulate form with an average particle size of less than 10
~m (or microns), most preferably 0.5 to 5 ~,m, for most effective delivery
to the distal lung.
Pharmaceutically acceptable carriers include carbohydrates such
15 as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other
ingredients for use in formulations may include DPPC, DOPE, DSPC
and DOPC. Natural or synthetic surfactants may be used. PEG may be
used (even apart from its use in derivatizing the protein or analog).
Dextrans, such as cyclodextran, may be used. Bile salts and other related
2o enhancers may be used. Cellulose and cellulose derivatives may be used.
Amino acids may be used, such as use in a buffer formulation.
Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is contemplated.
Formulations suitable for use with a nebulizer, either jet or
25 ultrasonic, will typically comprise the inventive compound dissolved in
water at a concentration of about 0.1 to 25 mg of biologically active protein
per mL of solution. The formulation may also include a buffer and a
simple sugar (e.g., for protein stabilization and regulation of osmotic
pressure). The nebulizer formulation may also contain a surfactant, to
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reduce or prevent surface induced aggregation of the protein caused by
atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the inventive
compound suspended in a propellant with the aid of a surfactant. The
propellant may be any conventional material employed for this purpose,
such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,
dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-
tetrafluoroethane, or combinations thereof. Suitable surfactants include
sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
Formulations for dispensing from a powder inhaler device will
comprise a finely divided dry powder containing the inventive compound
and may also include a bulking agent, such as lactose, sorbitol, sucrose,
rnannitol, trehalose, or xylitol in amounts which facilitate dispersal of the
powder from the device, e.g., 50 to 90% by weight of the formulation.
Nasal delivery forms. Nasal delivery of the inventive compound is
also contemplated. Nasal delivery allows the passage of the protein to the
2o blood stream directly after administering the therapeutic product to the
nose, without the necessity for deposition of the product in the lung.
Formulations for nasal delivery include those with dextran or
cyclodextran. Delivery via transport across other mucous membranes is
also contemplated.
Dosag-es. The dosage regimen involved in a method for treating the
above-described conditions will be determined by the attending physician,
considering various factors which modify the action of drugs, e.g. the age,
condition, body weight, sex and diet of the patient, the severity of any
infection,
time of administration and other clinical factors. Generally, the daily
regimen
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should be in the range of 0.1-1000 micrograms of the inventive compound per
kilogram of body weight, preferably 0.1-150 micrograms per kilogram.
Specific preferred embodiments
The inventors have determined preferred structures for the
preferred peptides listed in Table 4 below. The symbol "l1" may be any of
the linkers described herein or may simply represent a normal peptide
bond (i.e., so that no linker is present). Tandem repeats and linkers are
shown separated by dashes for clarity.
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Table 4-Preferred embodiments
Sequence/structure Peptide SEQ
descriptionID
NO:
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-A-F1PTH 161
1-34
SVSEIQLMHNRGKHLNSMERVEWLRKKLQDVHNF-A-F'(L11R) 162
PTH
1-34
SVSEIQLMHNKGKHLNSMERVEWLRKKLQDVHNF-A-F1(L11K) 163
PTH
1-34
SVSEIQLMHNLGKHLNSMRRVEWLRKKLQDVHNF-A-F1(E19R) 164
PTH
1-34
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV-A-F1PTH 165
1-31
SVSEIQLMHNLGKHLNSMERVEWLRKKLQD-A-F1 PTH 166
1-30
LLHNLGKSIQDLRRRFFLHHLIAEIHTA-A-F1 (D10N,K11L)167
PTHrP
7-34
SLALADDAAFRERARLLAALERRHWLNSY TIP39 168
MHKLLVLDAP-A-F1
PEG Cys27 PTH(1-169
34)
SVSEIQLMHNLGKHLNSMERVEWLRKCLQDVHNF
PEG Cys33 PTH(1-170
34)
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHCNF
PEG PEG Cys27, 171
33
PTH(1-34)
SVSEIQLMHNLGKHLNSMERVEWLRKCLQDVHCNF
"F1" is an Fc domain as defined previously herein or PEG, and "PEG" is a
molecule comprising polyethylene glycol as described previously herein
(e.g., mPEG, which is preferred), which may comprise any linker to enable
attachment of polyethylene glycol known in the art. In addition to those
listed in Table 4, the inventors further contemplate heterodimers in which
each strand of an Fc dimer is linked to a different peptide sequence; for
example, a molecule in which one strand can be described by SEQ ID NO:
166, the other by SEQ ID NO: 170 or an Fc linked with any of the
sequences in Tables 1A,1B, and 2.
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All of the compounds of this invention can be prepared by methods
described in (WO 00/24782).
The invention will now be further described by the following
working examples, which are illustrative rather than limiting.
Example 1
BIOACTIVITY OF AN Fc-CONJUGATED PTHIPTHrP RECEPTOR
(PTH-R1) AGONIST [PTH-(1-34)-Fc]
1o INTRODUCTION
Parathyroid hormone [PTH-(1-34) or native PTH-(1-84)] causes
increased bone formation and increased bone mass when injected daily.
This anabolic response was previously thought to require brief exposure to
PTH, which is facilitated by the short half-life (less than 1 h) of PTH.
15 Clinically, the anabolic effect of PTH therapy requires daily SC injection,
which is a significant barrier to the widespread use of PTH. Less frequent
injections of PTH would be clinically desirable and could be achieved by
increasing the in vivo half-life of PTH. Short-term (intermittent) exposure
to PTH (<1 h/day) stimulates osteoblastic bone formation, while long-
2o term (continuous) exposure (>2 h/day) stimulates osteoclastic bone
resorption (Dobnig et al, Endocrinolo~y 138: 4607,1998). The art suggests
that PTH with an extended half-life on its own may increase bone
resorption and lead to hypercalcemia. However, it should be possible to
prevent PTH-induced osteoclast activity with bone resorption inhibitors.
25 Osteoprotegerin (OPG) may be well suited for this purpose. A single
treatment of rats, mice or humans with OPG-Fc causes sustained
inhibition of bone resorption, by essentially eradicating the osteoclast
population. Co-administration of a potent bone resorption inhibitor, like
OPG, may provide greater effect. This regimen would theoretically permit
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the unopposed stimulation of bone formation by PTH, leading to
increased bone mass. It is likely that other bone resorption inhibitors,
including bisphosphonates or estrogen, would also inhibit PTH-induced
bone resorption and could therefore be used in combination with a long-
s acting PTH molecule. Towards this goal, we have cloned, expressed and
purified human PTH-(1-34)-Fc. Fc conjugation of proteins causes a
significant increase in their circulating half life, which may permit
injections of PTH-(1-34)-Fe on a schedule similar to or identical to that of
OPG-Fc. The benefits of this invention include less frequent injections of
1o PTH, from the current standard of once per day to as infrequently as once
per quarter.
MATERIALS, METHODS, AND RESULTS
Hypercalcemia Assay
15 We tested the potency and duration of effect of PTH-(1-34)-Fc in a
murine hypercalcemia model. Briefly, mice were injected once SC with
varying doses of PTH-(1-34) or PTH-(1-34)-Fc, and peripheral blood was
collected from the retroorbital sinus for determination of blood ionized
calcium. The half-life and the potency of PTH-(1-34)-Fc was greater than
2o that of PTH-(1-34), as evidenced by the sustained hypercalcemic response
of mice to the former agent (Figure 4). Hypercalcemia induced by PTH-(1-
34) persisted for 6-24 h, while equimolar doses of PTH-(1-34)-Fc caused
more sustained hypercalcernia (43-72 h). This duration of response is
consistent with greater half-life of the PTH-(1-34)-Fc construct vs. PTH-(1-
25 34). The potency of PTH-(1-34)-Fc was also significantly greater than that
of PTH-(1-34) (Figure 4). The highest dose of PTH-(1-34)-Fc caused a
greater increase in peak ionized calcium levels compared with an
equimolar dose of PTH-(1-34). Analysis of the area under the curve (ALTC)
demonstrated that at the highest dose employed, PTH-(1-34)-Fc caused a
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2.6-fold greater hypercalcemic response than did equimolar doses of PTH-
(1-34).
Anabolic Assay
Having demonstrated the superior pharmacology and half-life of
PTH-(1-34)-Fc over PTH-(1-34), we conducted a pilot study to determine
whether PTH-(1-34)-Fc co-treatment with OPG-Fc would increase bone
mass. Briefly, 6-month-old male Sprague Dawley (SD) rats were divided
into groups of 6. Baseline bone mineral density (BMD) was determined in
to the third lumbar vertebra (L3) of all rats by dual-energy X-ray
absorptiometry (DEXA) (Day 0). Rats were then treated according to the
following schedule:
Group 1: Vehicle controls (PBS, injected SC, Days 1, 3, and 5)
Group 2: OPG-Fc, single SC injection (1 mg/kg) on Day 1
Group 3: PTH-(1-34), SC injections on Days 1, 3, and 5, at 20
nMoles PTH/kg/injection. This represents an optimal anabolic PTH
regimen.
Group 4: Same as group 3, but with a single OPG-Fc injection on
Day 1.
2o Group 5: Single SC injection of PTH-(1-34)-Fc at 60 nMoles/kg, on
Day 1. This represents a molar dose which is equivalent to the total dose
of PTH-(1-34) received by group 3, but in a single injection.
Group 6: Same as group 5, but with a single OPG-Fc injection (SC,
1 mg/kg) on Day 1.
DEXA of the lumbar spine was performed again on Day 7 to
evaluate changes in BMD. BMD in L3 increased modestly with a single
injection of OPG-Fc, or with 3 injections of PTH-(1-34), compared to PBS-
treated rats (Figure 5). PTH-(1-34) + OPG caused a greater increase in
BMD than either OPG or PTH-(1-34) alone. As a monotherapy, a single
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injection of PTH-(1-34)-Fc failed to increase BMD. However, a single
injection of PTH-(1-34)-Fc plus a single injection of OPG-Fc caused a
significant increase in BMD (Figure 5). This result provides proof of
principle that a PTH construct with extended circulating half life can be
combined with a potent antiresorptive, like OPG-Fc, to create an anabolic
skeletal response. The anabolic effect of a single treatment with PTH-(1-
34)-Fc plus OPG-Fc was greater than that induced by multiple injections of
PTH-(1-34), with or without OPG-Fc co-treatment. In conclusion, maximal
gains in BMD can be achieved with infrequent injections of PTH-(1-34)-Fe
+ OPG-Fc, which is a superior treatment regimen compared to PTH-(1-34),
which must be injected daily or every second day.
Figure 5 shows the effect of PTH-Fc + OPG-Fc on bone mineral
density (BMD) in the third lumbar vertebra (L3). Normal 6 month old
male rats were treated with PTH-Fc or PTH or vehicle by a single SC
injection. Some rats also received a single SC injection of OPG. BMD was
determined 7 days later by DEXA. Data represent means ~ SD, n = 6
rats j group.
Example 2
2o BIOACTIVITY OF AN Fc-CONJUGATED PTH/PTHrP RECEPTOR
(PTH-R1) ANTAGONIST ([AsnlO,Leul1]PTHrP-(7-34)-Fc).
INTRODUCTION
Several disease states are associated with increased circulating
levels of PTH or PTHrP. Primary and secondary hyperparathyroidism
(PHPT and SHPT, respectively), axe associated with increased PTH levels,
while humoral hypercalcemia of malignancy (HHM) results in elevated
PTHrP levels. Both proteins signal through the common PTH/PTHrP
receptor (PTH-R1) to cause increases in bone resorption, renal calcium
3o reabsorption, and renal biosynthesis of vitamin D. While bone resorption
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inhibitors have variable success in inhibiting osteoclastic bone resorption
in these disease states, no therapy currently mitigates the intestinal and
renal influence of PTH or PTHrP excess on calcemia. Agents which
directly antagonize PTH or PTHrP signaling are therefore likely to have
greater efficacy compared to resorption inhibitors.
The most studied antagonists of PTH-R1 signaling are based on
amino terminal truncations. PTH-(7-34) peptides are fairly effective PTH-
R1 antagonists with very mild agonist activity. Compared to PTH-(7-34),
PTHrP-(7-34) peptide has greater affinity for PTH-R1 and as such is a
io more potent antagonist. However, PTHrP-(7-34) also has greater (but still
mild) agonist activity compared to PTH-(7-34) (McKee (1990), Endocrinol.
127: 76). The optimal antagonist may combine the weaker agonism of
PTH-(7-34) with the stronger antagonism of PTHrP-(7-34). Nutt et al
(1990), Endocrinol. 127: 491, demonstrates that substituting AsnlO and
Leul1 of PTH into the PTHrP sequence (replacing AsplO and Lysl1)
results in a peptide ([AsnlO,Leul1]PTHrP-(7-34)-Fc) with virtually no
agonist activity but with very potent antagonist activity.
Like native PTH, all peptide-based PTH-R1 antagonists share the
property of very short circulating half-lives (< 1 h). Furthermore, the
amino-terminal truncations which are required to remove receptor
agonism, also significantly reduce the affinity of these peptides for PTH-
R1. These properties limit the clinical potential of conventional peptide
antagonists. Fe-conjugation of amino-terminally truncated PTH- or PTHrP
peptides should significantly increase their circulating half life, such that
continuous antagonism of PTH-R1 might be achieved with sufficient
exposure to these Fc-antagonists.
MATERIALS, METHODS AND RESULTS
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We have cloned, expressed and purified [AsnlO,Leul1]PTHrP-(7-
34)-Fc. We tested the ability of this compound to antagonize both acute
and chronic hypercalcemia responses in mice. PTHrP-(1-34) was used as a
calcernic agent to evaluate the acute effects of [AsnlO,Leul1]PTHrP-(7-34)-
Fc. Because PTHrP is the principal mediator of HHM, this study also
represents a model for hypercalcemia-inducing tumors. Briefly, blood
ionized calcium (BIC) was measured at baseline, and mice were then
challenged with vehicle (PBS) or with PTHrP-(1-34) (0.5 mg/kg) by SC
injection. Mice were then treated once SC with varying doses of
[AsnlO,Leul1]PTHrP-(7-34)-Fc, or with vehicle (PBS). In vehicle-treated
mice challenged with PTHrP-(1-34), a transient hypercalcemic response
was observed. The peak calcemic response occurred at 3 h post challenge,
and persisted until at least 6 h post challenge. [AsnlO,Leul1]PTHrP-(7-
34)-Fc at 10 mg/kg caused a more rapid normalization of PTHrP-induced
hypercalcemia compared to vehicle treatment. A dose of 30 mg/kg
completely blocked the calcemic response to PTHrP-(1-34) (Figure 6).
In order to test the ability of [AsnlO,Leul1]PTHrP-(7-34)-Fc to
antagonize more chronic hypercalcemia, we used PTH-(1-34)-Fc as a long
acting calcemic agent. This study also represents a model for primary and
2o secondary hyperparathyroidism, diseases which are characterized by
persistent elevation of PTH levels. In vehicle-treated mice, a single SC
injection of PTH-(1-34)-Fc (30 mg/kg) caused a robust hypercalcemic
response in normal mice, reaching a level of 2.75 mg/dl at 24 h post
challenge (vs. normal control value of 1.35). A single SC injection of
[AsnlO,Leul1]PTHrP-(7-34)-Fc at 10-100 mg/kg caused a significant
decrease in the peak hypercalcemic response to PTH-(1-34)-Fc at 24 h
(Figure 6).
In conclusion, we have demonstrated antagonistic activity of
[AsnlO,Leul1]PTHrP-(7-34)-Fc, in both acute and chronic animal models
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of hypercalcemia. These models employed calcemic agents based on both
PTH and on PTHrP sequences. These data suggest that
[AsnlO,Leul1]PTHrP-(7-34)-Fc, as well as other Fc-conjugated PTH-R1
antagonists, may be effective treatment options for hyperparathyroidism,
HHM, and other diseases associated with aberrant PTH-R1 signaling.
Example 3
OSTEOGENIC PROPERTIES OF FC-CONJUGATED AND
NATIVE C-TERMINALLY TRUNCATED PTH FRAGMENTS
1o A. cAMP Assays
We tested the relative ability of PTH-Fc constructs to stimulate
cAIVIP accumulation in rat osteoblast-like ROS 17/2.8 cells. Cultures were
treated with the phosphodiesterase inhibitor IBMX to promote the
accumulation of CAMP. Cultures were then challenged for 15 minutes
15 with either vehicle (PBS), or various PTH constructs. Dose-dependent
cAMP accumulation was demonstrated for all fragments. Non-Fc-
conjugated PTH-(1-34) was slightly more potent than PTH-(1-31)-Fe and
PTH-(1-30)-Fc (Figure 7). These data demonstrate that Fc-conjugated PTH
fragments maintain the ability to activate the AC pathway in osteoblasts.
B. Mouse Bioassay
We then tested the effects of PTH-(1-34), PTH-(1-34)-Fc, PTH-(1-31)-Fc
and PTH-(1-30)-Fc in mice. Four week old male mice were injected on
days 0, 5, and 10 with vehicle or with PTH fragments, by SC injection.
Peripheral blood was obtained for clinical chemistry at 24, 48, and 72 h.
Mice were killed on day 15, vertebrae, tibiae and femurs were harvested
for histology and one tibia was collected for bone density measurements
(peripheral quantitative computed tomography, pQCT). Clinical
chemistry endpoints included total serum calcium, serum alkaline
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phosphatase (AP, a marker of osteoblast activity), and serum tartrate-
resistant acid phosphatase (TRAP, a marker of osteoclast activity). For
each animal, the ratio of AP:TRAP was calculated as an index of relative
osteoblast activity compared to osteoclast activity. A higher AP:TRAP
ratio would indicate a potentially more anabolic agent. The relatively high
doses (15-fold greater than optimal anabolic doses) were selected base on
previous studies which demonstrated significant changes in clinical
chemistry endpoints. It was anticipated that lower doses might be
required to demonstrate anabolic effects on bone density, and that
1o antiresorptive co-treatment might also be required to achieve anabolic
responses.
The clinical chemistry results are demonstrated in Figure 8. Serum
calcium was not significantly different at 24, 48, or 72 h after injection of
300 nmoles/kg (1.2 mg/kg) of PTH-(1-34). This result is consistent with
z5 the short half-life of the non-Fc conjugated peptide, which normally causes
a transient (12 h) increase in serum calcium. In contrast, an equimolar
dose of PTH-(1-34)-Fc caused a dramatic and sustained increase in serum
calcium, which peaked at 24 h. PTH-(1-31)-Fc was a more potent calcemic
agent, while PTH-(1-30)-Fc was the least calcemic of the 3 Fc peptides
20 (Figure 8A). Serum AP (osteoblast marker) was unchanged by PTH-(1-34)
administration, but was significantly elevated by 300 nmoles/kg of PTH-
(1-34)-Fc and by PTH-(1-31)-Fc at 72 h. PTH-(1-30)-Fc demonstrated the
greatest elevation of AP, which peaked 72 h after injection of 1,000
nmoles/kg (Figure 8B). Serum TRAP (osteoclast marker) was not
25 significantly changed by PTH-(1-34), PTH-(1-34)-Fc, or PTH-(1-30)-Fc, but
was dramatically increased by PTH-(1-31)-Fc (Figure 8C). The calculated
AP:TRAP ratios were unchanged by PTH-(1-34), and were increased over
time by PTH-(1-34)-Fc. The low dose of PTH-(1-31)-Fc (100 nmoles/kg)
increased AP:TRAP, while the high dose (1,000 nmoles/kg) decreased
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AP:TRAP. The greatest increase in AP:TRAP was realized with PTH-(1-
30)-Fc (1,000 nmoles/kg) (Figure SD).
The effects of the various PTH constructs on bone mineral density
(proximal tibial metaphysis) are demonstrated in Figure 9. At the end of
the 15-day study, PTH-(1-34) (300 nmoles/kg) was observed to have a
modest (non-significant) anabolic effect when injected on day 0, day 5 and
day 10. PTH-(1-34)-Fc (300 nmoles/kg) had no effect on bone density, nor
did PTH-(1-31)-Fc at 100 nmoles/kg. Higher doses of PTH-(1-31)-Fc (300-
1,000 nmoles/kg) caused significant hypercalcemia-related toxicity, and
1o bones were not harvested from these animals for pQCT. PTH-(1-30)-Fc
caused the greatest increase in bone density. There was an apparent
reverse dose-response, where PTH-(1-30)-Fc at 100 nmoles/kg had the
greatest effect and at 1,000 nmoles/kg had the least effect, although at all
doses BMD was greater than in controls (Figure 9). The reverse dose-
response was consistent with the notion that doses employed (chosen for
clinical chemistry endpoints) were 5-50 fold higher than the optimal
anabolic doses. Low doses of PTH (or PTH-Fc) which fail to significantly
increase serum calcium are optimal for anabolic effects. See Hock, J.M.
(1992), T. Bone Min. Res. 7:65-72. In the current study, the treatment
2o regimen with the greatest anabolic effect (PTH-(1-30)-Fc at 100 nmoles/kg)
was also the only PTH-Fe treatment which failed to significantly increase
serum calcium (Figure SA).
These data demonstrate the potential anabolic effects of C-
terminally truncated PTH-Fc peptides. The longer half life conferred by
Fc conjugation, combined with the selective stimulation of AC/cAMP by
C-terminal truncations, may explain the anabolic effect in the absence of a
potent bone resorption inhibitor. It is expected that stepwise C-terminal
truncation of PTH-(1-30)-Fc will reveal shorter fragments which maintain
or exceed the anabolic profile of PTH-(1-30)-Fc. These fragments may be
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more selective at stimulating osteoblasts, and may be less calcemic, thus
providing a wider therapeutic window for anabolic therapy.
Example 4
PTH-Fc TREATMENT AS A MONOTHERAPY
The efficacy of PTH-(1-34)-Fc as a monotherapy was demonstrated
in adult mice. Briefly, male BDF1 mice (4 months of age) were treated
twice per week by subcutaneous injection with various doses of PTH-(1-
34)-Fc or with vehicle (PBS). Other mice were treated daily with SC
1o injections of PTH-(1-34) at a dose of 80 ~g/kg/day (20 nmol/kg/day), a
treatment regimen which is optimal for increasing bone mass in rodents
(M. Gunness-Hey and J.M. Hock, Metab. Bone Dis. ~ Rel. Res. 5:177-181,
1984). After 3 weeks, mice were sacrificed and tibiae were analyzed for
bone mineral density (BMD) via pQCT (Figure 10).
Total tibial BMD and cancelled BMD were both significantly
increased by daily PTH-(1-34) injections compared to vehicle-treated
controls (Figure 1, two-way ANOVA, p<0.05). Twice-weekly injections of
PTH-(1-34)-Fc caused dose-dependent increases in both total and
cancellous BMD which, at the two highest doses (50 and 150 nmol/kg),
2o were significantly greater than the effects of either vehicle or daily PTH-
(1
34). Cortical BMD in the tibia was not significantly enhanced by daily
PTH-(1-34) treatments. Twice-weekly PTH-(1-34)-Fc caused a dose-
dependent increase in cortical BMD which at the highest dose was
signficantly greater than that observed in mice treated with vehicle or with
daily PTH-(1-34) (p<0.05).
Twice-weekly PTH-(1-34)-Fc also effectively increased BMD as a
monotherapy in aged ovariectomized (OVX) rats. Sprague Dawley rats
were OVX'd at 3 months of age and allowed to lose bone for 11 months.
Other rats were sham-operated and treated twice per week with vehicle
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(PBS). OVX rats were treated twice per week with SC injections of either
vehicle or the bisphosphonate APD (pamidronate, 0.5 mg/kg), or with
PTH-(1-34)-Fc (50 nmol/kg) or with APD + PTH-(1-34)-Fc. BMD was
determined weekly via dual energy X-ray absorptiometry (DEXA). Rats
were sacrificed after 4 weeks of treatment. At the start of treatment, OVX
rats had significant reductions in BMD at all skeletal sites analyzed,
compared to vehicle-treated OVX rats (Figure 11, p<0.05, 2-way ANOVA).
APD alone did not significantly increase BMD at any skeletal site
compared to vehicle-treated OVX rats. PTH-(1-34)-Fc alone caused a
1o significant increase in BMD at the femoral metaphysis after 4 weeks of
treatment (p<0.05). Treatment of OVX rats with PTH + ABD was
associated with an earlier significant increase in BMD at this site (3 weeks).
The combination of APD + PTH-(1-34)-Fc also caused significant BMD
increases at the lumbar vertebrae and at the femoral metaphysis (p<0.05).
PTH-(1-34)-Fc alone caused a mild and transient hypercalcemic response
which resolved spontaneously after day 10 despite continued treatments.
The co-administration of APD completely blocked the calcemic effect of
PTH-(1-34)-Fc.
These data suggest that PTH-(1-34)-Fc is an effective anabolic agent
2o when used as a monotherapy in both adult mice and aged OVX rats. We
have also demonstrated that the addition of an antiresorptive agent (APD)
to PTH-(1-34)-Fc was associated with similar or more rapid increases in
BMD in aged OVX rats. Co-administration of APD also blocked the
transient hypercalcemic response produced by PTH-(1-34)-Fc, which
suggests that the therapeutic index of PTH-(1-34)-Fc could be significantly
improved by co-administering an effective antiresorptive agent.
Example 5
PEG PTH ANALOGUES
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Cys mutations were introduced into PTH(1-34) to provide
PEGylation sites. The initial cysteine positions were selected based on
structure/function activity data suggesting that the C-terminal domain
may be most tolerant to PEGylation. These analogs represent a
substitution mutant K27C, an insertion mutant at C33 and the dual mutant
combining both cysteine positions:
PTH (1-34) analog (Cys 27) (SEQ ID NO: 188, appearing in Table 1B):
PTH (1-34) analog (Cys 33) (SEQ ID NO: 174, appearing in Table 1B):
PTH (1-34) analog (Cys 27/33) (SEQ ID N0:172, appearing in Table 1B):
1o Additional cysteine analogs of PTH (1-34) have also been prepared
(see Table 1B).
Chemical reagents. All Fmoc-amino acids and Fmoc-Phe-Wang
resin was obtained from Midwest Bio-Tech. (Fishers, IN). HOBt/DMF and
DCC/NMP were purchased from Q Bio-Gene (Carlsbad, CA). HBTU was
purchased from either Q Bio-Gene (Carlsbad, CA) or NovaBiochem (San
Diego, CA) Bulk solvents: ACN, MeOH, DCM, DMF, and NMP were
purchased from EM Science (Gibbstown, NJ). NEM and piperidine were
obtained from Aldrich (Milwaukee, WI). Phenol, and TIS were
purchased from Sigma (St. Louis, MO). TFA was purchased from either
2o PE Applied Biosystems (Foster City, CA) or Chem-Impex (Wood Dale, IL).
All reagents for amino acid analysis were purchased from PE Applied
Biosystems (Foster City, CA). All chemicals were of the highest grade
possible.
Solid-phase peptide synthesis of PTH(1-34) and analo ues. All
peptides were prepared by solid phase synthesis using the Fmoc/t-butyl-
based methodology with Fmoc-Phe-Wang or Fmoc-Cyc(Trt)-Wang resin
as a solid support. The sidechain protection scheme is as follows:
Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt),
Lys(Boc), Ser(tBu), and Trp(Boc). A PE Applied Biosystems (Foster City,
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CA) 431 instrument or a Rainin Symphony Multiplex Peptide Synthesizer
(Protein Technologies Inc, Woburn, MA) with the manufacturer's standard
Fmoc program was utilized for solid phase synthesis peptide chain
assembly at either 0.05, 0.10, or 0.20 mmol scale. Each coupling consisted
of the following modules: (i) removal of the a-amino Fmoc protection by
piperidine in NMP (ABI instrument) or NEM in DMF (Rainin instrument);
(ii) activation with either HBTU or DCC followed by (iii) single 60-minute
coupling of the HOBt ester of the Fmoc amino acid (20,10, or 5
equivalents) in NMP or DMF; (iv) resin washes.
1o Upon completion of the automated synthesis, the peptide-resin was
dried in vacuo and subjected to acidolytic deprotection and cleavage
(TFA:HzO:TIS:EDTahioanisol (85:5:5:2.5:2.5,10 ml, 4 hr, 20 °C). After
cleavage and deprotection, the resin/peptide suspension was filtered,
peptide-filtrate was precipitated using cold diethyl ether (40 ml), pelleted
by centrifugation, washed with cold diethyl ether (2x , 40 ml), and dried
in vacuo or by speedvac.
The precipitated crude peptides were analyzed by HPLC-MS for
crude purity and expected molecular ion. Analytical HPLC was carried
out on a Vydac (Hesperia, CA) 214TPTM C18 column (300 A pore size, 5~um
2o particle size, 0.46 x 25 cm). The conditions for this and all subsequent
primary analytical RP-HPLC were linear gradients of 5-50% ACN in 0.1%
aqueous TFA over 25 minutes, and a flow rate of 1.0 ml/min, unless
otherwise noted. The effluent was monitored with a PDA detector from
which the 220 nm absorbance profile was extracted.
Electrospray Mass Spectrometry. Mass spectra of all peptides were
obtained with an PE-Sciex API-I (Thornhill, ON) single quadrupole
electrospray mass spectrometer equipped with a nebulizer-assisted
electrospray source. Samples of preparative HPLC fractions were
introduced by means of an Alltech (Deerfield, IL) 426 HPLC pump and
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Perkin Elmer (Norwalk, CT) Series 200 autosampler, using a 0.40 ml/min
isocratic gradient of 1 mM NH40Ac in ACN:H20 (50:50). The mass
spectrometer was scanned from mass to charge (m/z) 300 to 2400 for the
characterization of the purified peptide. The acquisition time was 0.20
minutes, and 10 scans were used for each spectrum. Masses were
calculated from the m/z ratios from the observed protonation states of the
peptide using Sciex (Thornhill, ON) MacSpecTM software. Theoretical
masses were calculated using Sciex (Thornhill, ON) MacBioSpecTM
software
1o Secondary RP-HPLC anah~. All peptides were subjected to two
additional analytical RP-HPLC protocols to ensure peak homogeneity.
Samples were analyzed on a YMC (Wilmington, CA) ODS-AQ C18
column (2.0 x 50 mm). The conditions for this chromatography were a
linear gradient of 5 to 50% ACN in 0.1% aqueous TFA over 45 minutes,
z5 and a flow rate of 0.4 ml/min. Samples were also analyzed on a Vydac
(Hesperia, CA) 218TPTM C18 column (300 A. pore size, Sum particle size,
0.46 x 25 cm). The conditions for this analysis were a linear gradient of 5
to 20% ACN in 0.1% aqueous TFA over 10 minutes, followed by 20 to 50%
ACN in 0.1% aqueous TFA over 40 minutes, and a flow rate of 1 ml/min.
2o All secondary analytical Rl'-HPLC were monitored with a PDA detector
from which the 220 nm absorbance result was extracted.
Amino acid anah~. Qualitative amino acid analyses were
performed on purified peptides. All analyses were performed on an PE
Applied Biosystems (Foster City, CA) 420A derivatizer with a 130A
25 separation system. The peptides were manually hydrolyzed in vacuo
using 6 N HCl at 150 °C for 60 minutes and then applied (approximately
1
dug) to the amino acid analyzer for derivatization as the PTC derivative.
The amino acid mixture was then separated by RP-HPLC on a Brownlee
PTC C18 column (2.1 x 220 mm) with a linear gradient of 2% to 11% ACN
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in H20 over 4 minutes,11% to 27% ACN in H20 over 6 minutes, and 27%
to 47% ACN in H20 over 10 minutes. Both solvents were buffered with
NaOAc to pH 5.4, and the flow rate was 0.3 ml/min. Quantitative amino
acid analyses were performed in triplicate, and composition was
determined by comparison of each amino acid peak area in the peptide to
the area of a known amount of amino acid standard.
PEG lad tion. The PTH (1-34) cysteine analogs were PEGylated with
a variety of PEG-maleimides, as shown in Reaction Scheme 1 below.
0 0
~N ~'E~ Peptide-s
Peptide-sH -~- ~ \t~-PELT
Peptide cysteine analog O O
PEG-maleimide
PEG-Peptide conjugate
The PEGylation reactions were carried out in 20 mM sodium
phosphate, 5 mM EDTA, pH 6.5 with PTH (1-34) peptide concentrations
from 2-10 mg/ml and PEG:peptide stoichiometry of 0.5 to 5 fold molar
excess. Although reactant concentrations may exceed these limits, the
preferred conditions are 5 mg/ml peptide with an equimolar PEG-
maleimide concentration. Reaction times may range from 15 minutes to
overnight at room temperature or 4 degrees C, with 2 hours at room
temperature preferred. Optionally, the reaction may be stopped with the
addition of excess mercaptan, such as (3-mercaptoethanol. It is further
2o anticipated that PTH (1-34) PEGylation can be achieved by coupling in
non-aqueous solvents or through orthogonal approaches during peptide
synthesis. The PEG-PTH (1-34) conjugates were then purified by aqueous
phase cation exchange chromatography (Figure 13).
A list of PEGylated PTH peptides appears in Table 5 below
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Table 5--PEG-PTH peptides tested in vitro and in vivo. The
summarized assay results are relative to either PTH-(1-34) peptide or to
PTH-(1-34)-Fc, as indicated.
Peptide Name Description
C27 Peptide Analog, C27 mutant
C27-5K 5 kD, K27C Series
C27-10K 10 kD, K27C Series
C27-20K 201cD, K27C Series
C27-30K 30 kD, K27C Series
C27-2 X 20K 2 X 20 kD branched, K27C Series
C27-8K-C27 8 kD Dumbbell, K27C
C33 Peptide Analog, C33
C33-20K 20 kD, C33 Series
C27/33 Peptide Analog, C27/33
C27/33-2 X 20K 20 kD dual PEG, C27/33
C27/33-2 X 30K 30 kD dual, C27/33
C33-30K 30 kD, C33
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In vitro characterization by cAMP assay.PEG-PTH molecules were
screened in vitro for evidence of PTH receptor (PTHR1) activation by
monitoring production of cAMP in murine MC3T3-E1 osteoblast cultures.
PTH-(1-34) peptide consistently demonstrates the greatest potency in tlus
assay, while PTH-(1-34)-Fc is a full agonist with slightly reduced potency.
The mutant peptide analogs used for PEGylation (C27, C33 and C27/33)
had potencies in the cAMP assay that were similar to each other and to
PTH-(1-34)-Fc (Figure 14A through 14D). In most cases, PEGylation of
these peptides had little effect on their relative potency in cAMP assays,
1o and all PEG contructs appeared to behave as full agonists.
In vivo characterization: Hypercalcemia assay. PEG-PTH
molecules were also screened for evidence of sustained PTHR1 activation
in young male BDF1 mice. Briefly, miee were injected subcutaneously
with PEG-PTH or control constructs and blood ionized calcium (BIC)
15 levels were monitored for up to 120 hours. Increases in BIC reflect
increased bone resorption, which is a pharmacologic response to PTH
treatment. PTH-(1-34) peptide causes a transient hypercalcemic response
in mice which ends within about 6 hours of injection. In contrast, the
hypercalcemia induced by equimolar injections with PTH-(1-34)-Fc lasts
2o for approximately 72 hours (Figure 15). The longer duration of
hypercalcemia with PTH-Fc reflects a longer circulating half-life, an
attribute which also permits less frequent dosing relative to PTH peptide.
As expected, no hypercalcemic responses were evident at 24 hours after
injection of the non-PEGylated peptides PTH-(1-34), C27, or C33. This
25 observation is consistent with the predicted short half-life of these
peptides.
Other PEG-PTH constructs caused hypercalcemic responses which
were similar to those observed with equimolar doses of PTH-(1-34)-Fc,
including:
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~ C27-20K
~ C27-30K
~ C27-2 X 20K
~ C33-20K
The C27-30K construct caused the greatest hypercalcemic response
in this assay, and this peptide and C33-30K were then tested in a dose
response hypercalcemia assay. At equimolar doses, C27-30K and C33-30K
PEG-PTH molecules were 3-10 fold more potent than PTH-(1-34)-Fc in
hypercalcemia assays (Figure 16). For a given peak level of blood ionized
to calcium, therapeutic doses of PEG-PTH and PTH-Fc molecules caused
similar durations of hypercalcemia. At higher (toxic) doses, PEG-PTH
constructs caused a slightly longer duration of hypercalcemia relative to
PTH-(1-34)-Fc (Figure 16).
In vivo characterization: Bone anabolic assays. PEG-PTH
molecules were also tested for bone anabolic activity in adult male BDF1
mice. The doses were chosen based on the relative potencies of C27-30K
PEG-PTH and PTH-(1-34)-Fc in mouse hypercalcemia assays. PTH-(1-34)-
Fc at 150 nmol/kg caused a significant increase in tibial trabecular bone
mineral density (BMD) when injected twice-weekly for 4 weeks. C27-30K
2o PEG-PTH was similarly anabolic at 3-10 fold lower doses than PTH-(1-34)-
Fc when injected twice-weekly (Figure 17). C27-30K PEG-PTH also caused
significant gains in BMD when injected once weekly at the 50 nmol/kg
dose.
Discussion and conclusions. These data indicate the feasibility of
using PEGylation to increase the circulating half life of PTH peptides. The
longer half life conferred by PEGylation permits infrequent subcutaneous
dosing in rodents to obtain bone anabolic effects. The pharmacologic
effects of PEG-PTH are similar to those observed with PTH-Fe constructs,
with respect to cAMP activation in vitro as well as hypercalcemic and
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bone anabolic effects in vivo. These similarities in pharmacology suggest
that increasing the half-life of PTH peptides, regardless of the means,
could represent an important therapeutic advance. Neither Fc nor PEG
are absolutely required to realize the therapeutic benefit of a long-acting
PTH molecule. These data would also predict that a slow-release
formulation of PTH peptides such as those described in Tables 1A,1B and
2 would achieve the same desired benefit of less frequent injections.
The invention now being fully described, it will be apparent to one
to of ordinary skill in the art that many changes and modifications can be
made thereto, without departing from the spirit and scope of the invention
as set forth herein.
Abbreviations
as amino acid
AC adenylate cyclase
Acm acetamidomethyl
ACN acetonitrile
amu atomic mass unit
AP alkaline phosphatase
BMD bone mineral density
Boc tent-butyloxycarbonyl
(3-Me (3-mercaptoethanol
CAMP cyclic adenosine monophosphate
DCC Dicyclohexylcarbidiimide
DCM dichloromethane
Dde 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl
DEXA dual-energy X-ray absorptiometry
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DMF dimethyformamide
DTT dithiothreitol
EDT ethanedithiol
ES-MS electrospray-mass spectrometry
Fmoc fluorenylmethoxycarbonyl
HHM humoral hypercalcemia of malignancy
HOBt 1-hydroxybenzotriazole
HBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate;
to MeOH methanol
NEM N-ethylmorpholine
NH40ac ammonium acetate
NMP N methylpyrrolidinone
OPG osteoprotegerin
OV7C ovariectomized
PBS phosphate-buffered saline
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PDA photodiode array
pQCT peripheral quantitative computed tomography
2o PTC phenylisothiocyanate;
PTH parathyroid hormone
PTHrP parathyroid hormone-related protein
RP-HPLC reversed-phase high-pressure liquid chromatography
tBu tert butyl
TFA trifluoroacetic acid
TIS triisopropylsilane
TRAP tartrate-resistant acid phosphatase
Trt trityl (triphenylmethyl)
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