U.S. patent application number 13/686245 was filed with the patent office on 2013-03-28 for erythropoitein receptor peptide formulations and uses.
This patent application is currently assigned to AFFYMAX, INC.. The applicant listed for this patent is Affymax, Inc.. Invention is credited to Anne-Marie Duliege, Kerstin Leuther, Robert Barnett Naso, Richard Stead, Kathryn Wynne Woodburn.
Application Number | 20130079283 13/686245 |
Document ID | / |
Family ID | 40588748 |
Filed Date | 2013-03-28 |
United States Patent
Application |
20130079283 |
Kind Code |
A1 |
Duliege; Anne-Marie ; et
al. |
March 28, 2013 |
Erythropoitein Receptor Peptide Formulations and Uses
Abstract
The present invention relates to peptide compounds that are
agonists of the erythropoietin receptor (EPO-R). The invention also
relates to therapeutic methods using such peptide compounds to
treat disorders associated with insufficient or defective red blood
cell production. Pharmaceutical compositions, which comprise the
peptide compounds of the invention, and dosages are also
provided.
Inventors: |
Duliege; Anne-Marie; (Palo
Alto, CA) ; Stead; Richard; (Bellevue, WA) ;
Leuther; Kerstin; (San Jose, CA) ; Woodburn; Kathryn
Wynne; (Saratoga, CA) ; Naso; Robert Barnett;
(Menlo Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Affymax, Inc.; |
Palo Alto |
CA |
US |
|
|
Assignee: |
AFFYMAX, INC.
Palo Alto
CA
|
Family ID: |
40588748 |
Appl. No.: |
13/686245 |
Filed: |
November 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12178583 |
Jul 23, 2008 |
8324159 |
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13686245 |
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11777500 |
Jul 13, 2007 |
7919461 |
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12178583 |
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11446593 |
Jun 2, 2006 |
7550433 |
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11777500 |
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PCT/US2006/021845 |
Jun 5, 2006 |
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11777500 |
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60989758 |
Nov 21, 2007 |
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60957396 |
Aug 22, 2007 |
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60831049 |
Jul 14, 2006 |
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60687655 |
Jun 3, 2005 |
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60687655 |
Jun 3, 2005 |
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Current U.S.
Class: |
514/13.5 |
Current CPC
Class: |
C07K 17/08 20130101;
A61K 38/16 20130101 |
Class at
Publication: |
514/13.5 |
International
Class: |
C07K 17/08 20060101
C07K017/08 |
Claims
1. A method for treating a patient having a disorder characterized
by a deficiency of erythropoietin or a low or defective red blood
cell population, which method comprises administering to the
patient a therapeutically effective amount of a compound according
to Formula I that binds to and activates the erythropoietin
receptor (EPO-R), wherein the therapeutically effective amount is a
dosage of 0.025 to 0.5 milligram of the compound per 1 kilogram of
body weight of the patient ##STR00100## wherein PEG is a
polyethylene glycol moiety having a molecular weight of about
20,000 to about 50,000 Daltons.
2. The method according to claim 2 wherein the compound is Formula
Ia ##STR00101##
3. The method of claim 1, wherein the method further comprises a
pharmaceutically acceptable carrier.
4. The method of claim 1, wherein the disorder is renal failure or
dialysis.
5. The method of claim 4, wherein the therapeutically effective
amount is a dosage of 0.025 to 0.2 milligram of the compound per 1
kilogram of body weight of the patient.
6. The method of claim 5, wherein the dosage is 0.05 to 0.1
milligram of the compound per 1 kilogram of body weight of the
patient.
7. The method of claim 1, wherein the disorder is anemia associated
with a malignancy.
8. The method of claim 7, wherein the therapeutically effective
amount is a dosage of 0.075 to 0.5 milligram of the compound per 1
kilogram of body weight of the patient.
9. The method of claim 8, wherein the dosage is 0.2 to 0.4
milligram of the compound per 1 kilogram of body weight of the
patient.
10. The method of claim 1, wherein the therapeutically effective
amount is administered once every 3 to 4 weeks.
11. The method of claim 1, wherein the disorder is chronic kidney
disease.
12. The method of claim 11, wherein the therapeutically effective
amount is a dosage of 0.25 to 1.25 milligram of the compound per 1
kilogram of body weight of the patient.
13. The method of claim 12, wherein the dosage is 0.5 to 0.75
milligram of the compound per 1 kilogram of body weight of the
patient.
14. The method of claim 11, wherein the therapeutically effective
amount is administered once every 3 to 6 weeks.
15. The method of claim 14, wherein the therapeutically effective
amount is administered once every 4 weeks.
16. The method of claim 11, wherein the therapeutically effective
amount is administered by either intravenous or subcutaneous
injection.
17. The method of claim 16, wherein the therapeutically effective
amount is administered by subcutaneous injection.
18. The method of claim 7, wherein the therapeutically effective
amount is a dosage of 0.05 to 0.5 milligram of the compound per 1
kilogram of body weight of the patient.
19. The method of claim 7, wherein the malignancy is solid tumor
malignancy or lymphoma.
Description
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 60/989,758
filed Nov. 21, 2007 and to U.S. Provisional Patent Application No.
60/957,396 filed Aug. 22, 2007. This application is also a
continuation-in-part of pending each of which claims priority to
U.S. patent application Ser. No. 11/777,500 filed Jul. 13, 2007,
which claims priority to U.S. Provisional Application No.
60/831,049 filed Jul. 14, 2006. U.S. patent application Ser. No.
11/777,500 is a continuation-in-part of U.S. patent application
Ser. No. 11/446,593 filed Jun. 2, 2006 and International Patent
Application No. PCT/US2006/021845 filed on Jun. 5, 2006, both of
which claim priority to U.S. Provisional to Patent Application Ser.
No. 60/687,655 filed on Jun. 3, 2005. The contents of all of the
above-cited applications are incorporated into the present
disclosure by reference and in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to peptide compounds that are
agonists of the erythropoietin receptor (EPO-R). The invention also
relates to therapeutic methods using such peptide compounds to
treat disorders associated with insufficient or defective red blood
cell production. Pharmaceutical compositions, which comprise the
peptide compounds of the invention, are also provided.
BACKGROUND OF THE INVENTION
[0003] Erythropoietin (EPO) is a glycoprotein hormone of 165 amino
acids, with a molecular weight of about 34 kilodaltons (kD) and
preferred glycosylation sites on amino-acid positions 24, 38, 83,
and 126. It is initially produced as a precursor protein with a
signal peptide of 23 amino acids. EPO can occur in three forms:
.alpha., .beta., and asialo. The .alpha. and .beta. forms differ
slightly in their carbohydrate components, but have the same
potency, biological activity, and molecular weight. The asialo form
is an .alpha. or .alpha. form with the terminal carbohydrate
(sialic acid) removed. The DNA sequences encoding EPO have been
reported [U.S. Pat. No. 4,703,008 to Lin].
[0004] EPO stimulates mitotic division and differentiation of
erythrocyte precursor cells, and thus ensures the production of
erythrocytes. It is produced in the kidney when hypoxic conditions
prevail. During EPO-induced differentiation of erythrocyte
precursor cells, globin synthesis is induced; heme complex
synthesis is stimulated; and the number of ferritin receptors
increases. These changes allow the cell to take on more iron and
synthesize functional hemoglobin, which in mature erythrocytes
binds oxygen. Thus, erythrocytes and their hemoglobin play a key
role in supplying the body with oxygen. These changes are initiated
by the interaction of EPO with an appropriate receptor on the cell
surface of the erythrocyte precursor cells [See, e.g., Graber and
Krantz (1978) Ann. Rev. Med. 29.51-66].
[0005] EPO is present in very low concentrations in plasma when the
body is in a healthy state wherein tissues receive sufficient
oxygenation from the existing number of erythrocytes. This normal
low concentration is sufficient to stimulate replacement of red
blood cells which are lost normally through aging.
[0006] The amount of EPO in the circulation is increased under
conditions of hypoxia when oxygen transport by blood cells in the
circulation is reduced. Hypoxia may be caused, for example, by
substantial blood loss through hemorrhage, destruction of red blood
cells by over-exposure to radiation, reduction in oxygen intake due
to high altitude or prolonged unconsciousness, or various forms of
anemia. In response to such hypoxic stress, elevated EPO levels
increase red blood cell production by stimulating the proliferation
of erythroid progenitor cells. When the number of red blood cells
in circulation is greater than needed for normal tissue oxygen
requirements, EPO levels in circulation are decreased.
[0007] Because EPO is essential in the process of red blood cell
formation, this hormone has potentially useful applications in both
the diagnosis and the treatment of blood disorders characterized by
low or defective red blood cell production. Recent studies have
provided a basis for the projection of EPO therapy efficacy for a
variety of disease states, disorders, and states of hematologic
irregularity, including: beta-thalassemia [see, Vedovato, et al.
(1984) Acta. Haematol. 71:211-213]; cystic fibrosis [see,
Vichinsky, et al. (1984) J. Pediatric 105:15-21]; pregnancy and
menstrual disorders [see, Cotes, et al. (193) Brit. J. Ostet.
Gyneacol. 90:304-311]; early anemia of prematurity [see, Haga, et
al. (1983) Acta Pediatr. Scand. 72; 827-831]; spinal cord injury
[see, Claus-Walker, et al. (1984) Arch. Phys. Med. Rehabil.
65:370-374]; space flight [see, Dunn, et al. (1984) Eur. J. Appl.
Physiol. 52:178-182]; acute blood loss [see, Miller, et al. (1982)
Brit. J. Haematol. 52:545-590]; aging [see, Udupa, et al. (1984) J.
Lab. Clin. Med. 103:574-580 and 581-588 and Lipschitz, et al.
(1983) Blood 63:502-509]; various neoplastic disease states
accompanied by abnormal erythropoiesis [see, Dainiak, et al. (1983)
Cancer 5:1101-1106 and Schwartz, et al. (1983) Otolaryngol.
109:269-272]; and renal insufficiency [see, Eschbach. et al. (1987)
N. Eng. J. Med. 316:73-78].
[0008] Purified, homogeneous EPO has been characterized [U.S. Pat.
No. 4,677,195 to Hewick]. A DNA sequence encoding EPO was purified,
cloned, and expressed to produce recombinant polypeptides with the
same biochemical and immunological properties and natural EPO. A
recombinant EPO molecule with oligosaccharides identical to those
on natural EPO has also been produced [See, Sasaki, et al. (1987)
J. Biol. Chem. 262:12059-12076].
[0009] The biological effect of EPO appears to be mediated, in
part, through interaction with a cell membrane bound receptor.
Initial studies, using immature erythroid cells isolated from mouse
spleen, suggested that the EPO-binding cell surface proteins
comprise two polypeptides having approximate molecular weights of
85,000 Daltons and 100,000 Daltons, respectively [Sawyer, et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3690-3694]. The number of
EPO-binding sites was calculated to average from 800 to 1000 per
cell surface. Of these binding sites, approximately 300 bound EPO
with a K.sub.d of approximately 90 pM (picomolar), while the
remaining bound EPO with a reduced affinity of approximately 570 pM
[Sawyer, et al. (1987) J. Biol. Chem. 262:5554-5562]. An
independent study suggested that EPO-responsive splenic
erythroblasts, prepared from mice injected with the anemic strain
(FVA) of the Friend leukemia virus, possess a total of
approximately 400 high and low affinity EPO binding sites with
K.sub.d values of approximately 100 pM and 800 pM, respectively
[Landschulz, et al. (1989) Blood 73:1476-1486].
[0010] Subsequent work indicated that the two forms of EPO receptor
(EPO-R) were encoded by a single gene. This gene has been cloned
[See, e.g., Jones, et al. (1990) Blood 76, 31-35; Noguchi, et al.
(1991) Blood 78:2548-2556; Maouche, et al. (1991) Blood
78:2557-2563]. For example, the DNA sequences and encoded peptide
sequences for murine and human EPO-R proteins are described in PCT
Pub. No. WO 90/08822 to D'Andrea, et al. Current models suggest
that binding of EPO to EPO-R results in the dimerization and
activation of two EPO-R molecules, which results in subsequent
steps of signal transduction [See, e.g., Watowich, et al. (1992)
Proc. Natl. Acad. Sci. USA 89:2140-2144].
[0011] The availability of cloned genes for EPO-R facilitates the
search for agonists and antagonists of this important receptor. The
availability of the recombinant receptor protein allows the study
of receptor-ligand interaction in a variety of random and
semi-random peptide diversity generation systems. These systems
include the "peptides on plasmids" system [described in U.S. Pat.
No. 6,270,170]; the "peptides on phage" system [described in U.S.
Pat. No. 5,432,018 and Cwirla, et al. (1990) Proc. Natl. Acad. Sci.
USA 87:6378-6382]; the "encoded synthetic library" (ESL) system
[described in U.S. patent application Ser. No. 946,239, filed Sep.
16, 1992]; and the "very large scale immobilized polymer synthesis"
system [described in U.S. Pat. No. 5,143,854; PCT Pub. No.
90/15070; Fodor, et al. (1991) Science 251:767-773; Dower and Fodor
(1991) Ann. Rep. Med. Chem. 26:271-180; and U.S. Pat. No.
5,424,186].
[0012] Peptides that interact to a least some extent with EPO-R
have been identified and are described, for example in U.S. Pat.
Nos. 5,773,569; 5,830,851; and 5,986,047 to Wrighton, et al.; PCT
Pub. No. WO 96/40749 to Wrighton, et al.; U.S. Pat. No. 5,767,078
and PCT Pub. No. 96/40772 to Johnson and Zivin; PCT Pub. No. WO
01/38342 to Balu; and WO 01/91780 to Smith-Swintosky, et al. In
particular, a group of peptides containing a peptide motif has been
identified, members of which bind to EPO-R and stimulate
EPO-dependent cell proliferation. Yet, peptides identified to date
that contain the motif stimulate EPO-dependent cell proliferation
in vitro with EC50 values of about 20 nanomolar (nM) to about 250
nM. Thus, peptide concentrations of 20 nM to 250 nM are required to
stimulate 50% of the maximal cell proliferation stimulated by EPO.
Still other peptides and constructs thereof that bind to the EPO
receptor have been described in U.S. provisional application Ser.
Nos. 60/470,244, 60/470,245, and 60/469,993, all filed on May 12,
2003; U.S. provisional application Ser. Nos. 60/627,432 and
60/627,433 both filed on Nov. 11, 2004; U.S. non-provisional
application Ser. No. 10/844,968, filed on May 12, 2004; and
International Application Serial Nos. PCT/US2004/14886 and
PCT/US2004/014889, both filed on May 12, 2004, and published as WO
2004/101611 and WO 2004/101606, respectively. Each of these
applications is hereby incorporated by reference and in its
entirety.
[0013] Given the immense potential of EPO-R agonists, both for
studies of the important biological activities mediated by this
receptor and for treatment of disease, there remains a need for the
identification of peptide EPO-R agonists of enhanced potency and
activity. The present invention provides such compounds.
[0014] The citation and/or discussion of cited references in this
section and throughout the specification is provided merely to
clarify the description of the present invention and is not an
admission that any such reference is "prior art" to the present
invention.
SUMMARY OF THE INVENTION
[0015] The present invention provides novel peptide compounds,
which are EPO-R agonists of dramatically enhanced potency and
activity. These peptide compounds are homodimers of peptide
monomers having the amino acid sequence
(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1), or homodimers of
peptide monomers having the amino acid sequence
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2), homodimers of
peptide monomers having the amino acid sequence
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG) (SEQ ID NO: 3); where each amino
acid is indicated by standard one letter abbreviation, "(AcG)" is
N-acetylglycine, "(1-nal)" is 1-naphthylalanine, and "(MeG)" is
N-methylglycine, also known as sarcosine. Each peptide monomer of a
peptide dimer contains an intramolecular disulfide bond between the
cysteine residues of the monomer.
[0016] The peptide monomers may be dimerized by covalent attachment
to a branched tertiary amide linker. The tertiary amide linker can
be depicted as:
--C.sup.1O--CH.sub.2--X--CH.sub.2--C.sup.2O
where: X is NCO--(CH.sub.2).sub.2--N.sup.1H--; C.sup.1 of the
linker forms an amide bond with the .epsilon.-amino group of the
C-terminal lysine residue of the first peptide monomer; C.sup.2 of
the linker forms an amide bond with the .epsilon.-amino group of
the C-terminal lysine residue of the second peptide monomer; and
N.sup.1 of X is attached via a carbamate linkage or an amide
linkage to an activated polyethylene glycol (PEG) moiety, where the
PEG has a molecular weight of about 20,000 to about 40,000 Daltons
(the term "about" indicating that in preparations of PEG, some
molecules will weigh more, some less, than the stated molecular
weight).
[0017] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N.sup.1
of the linker is attached via a carbamate linkage to an activated
polyethylene glycol (PEG) moiety, the novel peptide compounds of
the invention may be represented as follows:
##STR00001##
[0018] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N.sup.1
of the linker is attached via an amide linkage to an activated
polyethylene glycol (PEG) moiety, the novel peptide compounds of
the invention may be represented as follows:
##STR00002##
Where each monomer of the homodimer has the amino acid sequence,
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and N.sup.1 of
the linker is attached via a carbamate linkage to an activated
polyethylene glycol (PEG) moiety, the novel peptide compounds of
the invention may be represented as follows:
##STR00003##
[0019] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
N.sup.1 of the linker is attached via an amide linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00004##
[0020] The peptide monomers may also be dimerized by covalent
attachment to a branched tertiary amide linker. The tertiary amide
linker can be depicted as:
--C.sup.1O--CH.sub.2--X--CH.sub.2--C.sup.2O--
where: X is NCO--(CH.sub.2).sub.2--NH--C.sup.3O--; C.sup.1 of the
linker forms an amide bond with the .epsilon.-amino group of the
C-terminal lysine residue of the first peptide monomer; and C.sup.2
of the linker forms an amide bond with the .epsilon.-amino group of
the C-terminal lysine residue of the second peptide monomer. The
peptide dimers of the invention further comprise a spacer moiety of
the following structure:
--N.sup.1H--(CH.sub.2).sub.4--C.sup.4H--N.sup.2H--
where: C.sup.4 of the spacer is covalently bonded to C.sup.3 of X;
N.sup.1 of the spacer is covalently attached via a carbamate or an
amide linkage to an activated polyethylene glycol (PEG) moiety; and
N.sup.2 of the spacer is covalently attached via a carbamate or an
amide linkage to an activated PEG moiety, where PEG has a molecular
weight of about 10,000 to about 50,000 Daltons (the term "about"
indicating that in preparations of PEG, some molecules will weigh
more, some less, than the stated molecular weight). Each PEG moiety
may be, individually, 10,000 Daltons (10 kD), 20 kD, 30 kD, 40 kD,
or 50 kD.
[0021] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both
N.sup.1 and N.sup.2 of the spacer are covalently attached via a
carbamate linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00005##
In preferred embodiments, the C-terminal lysine of the two peptide
monomers is L-lysine. Also, those skilled in the art will
appreciate from the above chemical structures that the two linear
PEG moieties are joined by lysine (e.g., as mPEG.sub.2-Lys-NHS or
as mPEG.sub.2-Lysinol-NPC), which is also preferably L-lysine and
giving rise to the following stereochemistry.
##STR00006##
[0022] Alternatively, one or more of the lysine residues can be a
D-lysine, giving rise to alternative stereochemistries which will
be readily appreciate by those skilled in the art.
[0023] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both
N.sup.1 and N.sup.2 of the spacer are covalently attached via an
amide linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented, as follows:
##STR00007##
Again, the lysine molecules in this compound are preferably all
L-lysine, giving rise to the following stereochemistry.
##STR00008##
Alternatively, one or more of the lysine residues can be a
D-lysine, giving rise to alternative stereochemistries which will
be readily appreciated by those skilled in the art.
[0024] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
both N.sup.1 and N.sup.2 of the spacer are covalently attached via
a carbamate linkage to an activated PEG moiety, wherein Y is a
carbamate group, the novel peptide compounds of the invention may
be represented as follows:
##STR00009##
Preferably, the lysine residues joining the peptide monomer and
linear PEG moieties in this molecule are all L-lysine, giving rise
to the following stereochemistry:
##STR00010##
[0025] Alternatively, one or more of the lysine residues can be a
D-lysine, giving rise to alternative stereochemistries that will be
readily appreciated by those of ordinary skill in the art.
[0026] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
both N.sup.1 and N.sup.2 of the spacer are covalently attached via
an amide linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00011##
Preferably, the lysine residues joining the peptide monomer and
linear PEG moieties of this molecule are all L-lysine, giving rise
to the following stereochemistry.
##STR00012##
In other embodiments, one or more of the lysine residues can be a
D-lysine, giving rise to alternative stereochemistries that will be
readily appreciated by persons of ordinary skill in the art.
[0027] The peptide monomers may also be dimerized by attachment to
a lysine linker, whereby one peptide monomer is attached at its
C-terminus to the lysine's .epsilon.-amino group and the second
peptide monomer is attached at its C-terminus to the lysine's
.alpha.-amino group.
[0028] The peptide dimers of the invention further comprise a
spacer moiety of the following structure:
--N.sup.1H--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2---
N.sup.2H--
At one end, N.sup.1 of the spacer is attached via an amide linkage
to a carbonyl carbon of the lysine linker. At the opposite end,
N.sup.2 of the spacer is attached via a carbamate linkage or an
amide linkage to an activated polyethylene glycol (PEG) moiety,
where the PEG has a molecular weight of about 20,000 to about
40,000 Daltons (the term "about" indicating that in preparations of
PEG, some molecules will weigh more, some less, than the stated
molecular weight).
[0029] Where the spacer is attached via a carbamate linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention (SEQ ID NO: 3) may be represented as
follows:
##STR00013##
[0030] Where the spacer is attached via an amide linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention (SEQ ID NO: 3) may be represented as
follows:
##STR00014##
[0031] The invention further provides methods to treat various
medical conditions using such peptide compounds. The methods
include treating a patient having a disorder characterized by a
deficiency of erythropoietin or a low or defective red blood cell
population by administering to the patient a therapeutically
effective amount of one of the above compounds. In certain
embodiments, the disorder is end stage renal failure or dialysis;
chronic kidney disease, anemia associated with AIDS, auto immune
disease or a malignancy; beta-thalassemia; cystic fibrosis; early
anemia of prematurity; anemia associated with chronic inflammatory
disease; spinal cord injury; acute blood loss; aging; or neoplastic
disease states accompanied by abnormal erythropoiesis. Furthermore,
in certain embodiments, the disorder is renal failure or dialysis,
and the therapeutically effective amount is a dosage of 0.025 to
0.2 milligram of the compound per 1 kilogram body weight of the
patient. In other embodiments, the disorder is renal failure or
dialysis, and the therapeutically effective amount is a dosage of
0.05 to 0.1 milligram of the compound per 1 kilogram body weight of
the patient. In certain embodiments, the disorder is anemia
associated with a malignancy, and the therapeutically effective
amount is a dosage of 0.05 to 0.5 milligram of the compound per 1
kilogram body weight of the patient, or alternatively, between
0.075 to 0.5 milligram of the compound per 1 kilogram body weight
of the patient. In other embodiments, the disorder is anemia
associated with a malignancy, and the therapeutically effective
amount is a dosage is 0.2 to 0.4 milligram of the compound per 1
kilogram body weight of the patient. The therapeutically effective
amount can be administered once every 3 to 4 weeks.
[0032] In preferred embodiments, the disorder is chronic kidney
disease and the therapeutically effective amount is preferably a
dosage of 0.25 to 1.25 milligram of the compound per 1 kilogram of
body weight of the patient, more preferably the dosage is 0.5 to
0.75 milligram of the compound per 1 kilogram of body weight of the
patient. The therapeutically effective amount can be administered
once every 3 to 6 weeks. In preferred embodiments, the
therapeutically effective amount is administered once every 4
weeks. The therapeutically effective amount can be administered by
either intravenous or subcutaneous injection. In preferred
embodiments, the therapeutically effective amount is administered
by subcutaneous injection. In particularly preferred embodiments
the chronic kidney disease patient is pre-dialysis.
[0033] The invention further provides pharmaceutical compositions
comprised of such peptide compounds. In certain embodiments, the
PEG has a molecular weight of about 20,000 Daltons. In other
embodiments, the pharmaceutical composition comprises any one of
the above compounds and a pharmaceutically acceptable carrier.
DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 depicts the mean reticulocyte change from baseline
for 0-12 weeks.
[0035] FIG. 2 depicts the mean hemoglobin (Hgb) change from
baseline for 0-12 weeks.
[0036] FIG. 3 depicts correction of anemia (Hgb>11 g/dL) by dose
and treatment duration.
[0037] FIG. 4 depicts the mean hemoglobin (Hgb) change from
baseline for 12-22 weeks.
[0038] FIG. 5 depicts the hemoglobin (Hgb) change from baseline for
individual patients in the 0.05 mg/kg cohort for 0 to 12 weeks.
[0039] FIG. 6 depicts the percent of pateints with a hemoglobin
increase of .gtoreq.1 g/dL from baseline for individual patients in
the 0.5 mg/kg, 0.1 mg/kg, 0.15 mg/kg, and 0.2 mg/kg cohort for 3,
6, 9, and 12 weeks.
[0040] FIG. 7 depicts the percent of patients showing a hemoglobin
increase of .gtoreq.2 g/dL or the combination of a .gtoreq.1 g/dL
increase and a hemoglobin value of at least 11 g/dL in the 0.5
mg/kg, 0.1 mg/kg, 0.15 mg/kg, and 0.2 mg/kg dose cohorts.
[0041] FIG. 8 depicts hemoglobin increases from baseline for
patients in the 0.1 mg/kg, 0.15 mg/kg, and 0.2 mg/kg dose cohorts
for 0 to 14 weeks.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0042] Amino acid residues in peptides are abbreviated as follows:
Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile
or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or
S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q;
Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or
D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is
Trp or W; Arginine is Arg or R; and Glycine is Gly or G. The
unconventional amino acids in peptides are abbreviated as follows:
1-naphthylalanine is 1-nal or Np; N-methylglycine (also known as
sarcosine) is MeG or Sc; and acetylated glycine (N-acetylglycine)
is AcG.
[0043] As used herein, the term "polypeptide" or "protein" refers
to a polymer of amino acid monomers that are alpha amino acids
joined together through amide bonds. Polypeptides are therefore at
least two amino acid residues in length, and are usually longer.
Generally, the term "peptide" refers to a polypeptide that is only
a few amino acid residues in length. The novel EPO-R agonist
peptides of the present invention are preferably no more than about
50 amino acid residues in length. They are more preferably of about
17 to about 40 amino acid residues in length. A polypeptide, in
contrast with a peptide, may comprise any number of amino acid
residues. Hence, the term polypeptide included peptides as well as
longer sequences of amino acids.
[0044] As used herein, the phrase "pharmaceutically acceptable"
refers to molecular entities and compositions that are "generally
regarded as safe", e.g., that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin.
[0045] As used herein the term "agonist" refers to a biologically
active ligand which binds to its complementary biologically active
receptor and activates the latter either to cause a biological
response in the receptor, or to enhance preexisting biological
activity of the receptor.
Novel Peptides that are EPO-R Agonists
[0046] The present invention provides novel peptide compounds,
which are EPO-R agonists of dramatically enhanced potency and
activity. These peptide compounds are homodimers of peptide
monomers having the amino acid sequence
(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1), or homodimers of
peptide monomers having the amino acid sequence
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2); where each
amino acid is indicated by standard one letter abbreviation,
"(AcG)" is N-acetylglycine, "(1-nal)" is 1-naphthylalanine, and
"(MeG)" is "(MeG)" is N-methylglycine, also known as sarcosine.
Each peptide monomer of a peptide dimer contains an intramolecular
disulfide bond between the cysteine residues of the monomer. Such
monomers may be represented schematically as follows:
##STR00015##
[0047] These monomeric peptides are dimerized to provide peptide
dimers of enhanced EPO-R agonist activity. The linker (L.sub.K)
moiety is a branched tertiary amide, which bridges the C-termini of
two peptide monomers, by simultaneous attachment to the C-terminal
lysine residue of each monomer. The tertiary amide linker can be
depicted as:
--C.sup.1O--CH.sub.2--X--CH.sub.2--C.sup.2O--
where: X is NCO--(CH.sub.2).sub.2--N.sup.1H--; C.sup.1 of the
linker forms an amide bond with the .epsilon.-amino group of the
C-terminal lysine residue of the first peptide monomer; C.sup.2 of
the linker forms an amide bond with the .epsilon.-amino group of
the C-terminal lysine residue of the second peptide monomer; and
N.sup.1 of X is attached via a carbamate linkage or an amide
linkage to an activated polyethylene glycol (PEG) moiety, where the
PEG has a molecular weight of about 20,000 to about 40,000 Daltons
(the term "about" indicating that in preparations of PEG, some
molecules will weigh more, some less, than the stated molecular
weight).
[0048] The tertiary amide linker may also be depicted as:
--C.sup.1O--CH.sub.2--X--CH.sub.2--C.sup.2O--
where: X is NCO--(CH.sub.2).sub.2--NH--C.sup.3O--; C.sup.1 of the
linker forms an amide bond with the .epsilon.-amino group of the
C-terminal lysine residue of the first peptide monomer; and C.sup.2
of the linker forms an amide bond with the .epsilon.-amino group of
the C-terminal lysine residue of the second peptide monomer. The
peptide dimers of the invention further comprise a spacer moiety of
the following structure:
[0049] --N.sup.1H--(CH.sub.2).sub.4--C.sup.4H--N.sup.2H--
where: C.sup.4 of the spacer is covalently bonded to C.sup.3 of X;
N.sup.1 of the spacer is covalently attached via a carbamate or an
amide linkage to an activated PEG moiety; and N.sup.2 of the spacer
is covalently attached via a carbamate or an amide linkage to an
activated PEG moiety, where PEG has a molecular weight of about
10,000 to about 60,000 Daltons (the term "about" indicating that in
preparations of PEG, some molecules will weigh more, some less,
than the stated molecular weight).
[0050] Thus, the novel peptides of the invention can also contain a
PEG moiety, which is covalently attached via a carbamate linkage or
an amide linkage to the tertiary amide linker of the peptide dimer.
PEG is a water soluble polymer that is pharmaceutically acceptable.
PEG for use in the present invention may be linear, unbranched PEG
having a molecular weight of about 20 kilodaltons (20K) to about
60K (the term "about" indicating that in preparations of PEG, some
molecules will weigh more, some less, than the stated molecular
weight). Most preferably, the PEG has a molecular weight of about
30K to about 40K. One skilled in the art will be able to select the
desired polymer size based on such considerations as the desired
dosage; circulation time; resistance to proteolysis; effects, if
any, on biological activity; ease in handling; degree or lack of
antigenicity; and other known effects of PEG on a therapeutic
peptide.
[0051] Peptides, peptide dimers and other peptide-based molecules
of the invention can be attached to water-soluble polymers (e.g.,
PEG) using any of a variety of chemistries to link the
water-soluble polymer(s) to the receptor-binding portion of the
molecule (e.g., peptide+spacer). A typical embodiment employs a
single attachment junction for covalent attachment of the water
soluble polymer(s) to the receptor-binding portion, however in
alternative embodiments multiple attachment junctions may be used,
including further variations wherein different species of
water-soluble polymer are attached to the receptor-binding portion
at distinct attachment junctions, which may include covalent
attachment junction(s) to the spacer and/or to one or both peptide
chains. In some embodiments, the dimer or higher order multimer
will comprise distinct species of peptide chain (i.e., a
heterodimer or other heteromultimer). By way of example and not
limitation, a dimer may comprise a first peptide chain having a PEG
attachment junction and the second peptide chain may either lack a
PEG attachment junction or utilize a different linkage chemistry
than the first peptide chain and in some variations the spacer may
contain or lack a PEG attachment junction and said spacer, if
PEGylated, may utilize a linkage chemistry different than that of
the first and/or second peptide chains. An alternative embodiment
employs a PEG attached to the spacer portion of the
receptor-binding portion and a different water-soluble polymer
(e.g., a carbohydrate) conjugated to a side chain of one of the
amino acids of the peptide portion of the molecule.
[0052] A wide variety of polyethylene glycol (PEG) species may be
used for PEGylation of the receptor-binding portion
(peptides+spacer). Substantially any suitable reactive PEG reagent
can be used. In preferred embodiments, the reactive PEG reagent
will result in formation of a carbamate or amide bond upon
conjugation to the receptor-binding portion. Suitable reactive PEG
species include, but are not limited to, those which are available
for sale in the Drug Delivery Systems catalog (2003) of NOF
Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome,
Shibuya-ku, Tokyo 150-6019) and the Molecular Engineering catalog
(2003) of Nektar Therapeutics (490 Discovery Drive, Huntsville,
Ala. 35806). For example and not limitation, the following PEG
reagents are often preferred in various embodiments: mPEG2-NHS,
mPEG2-ALD, multi-Arm PEG, mPEG(MAL)2, mPEG2(MAL), mPEG-NH12,
mPEG-SPA, mPEG-SBA, mPEG-thioesters, mPEG-Double Esters, mPEG-BTC,
mPEG-ButyrALD, mPEG-ACET, heterofunctional PEGs (NH2-PEG-COOH,
Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates
(ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE), multiarmed
PEGs of the SUNBRITE series including the GL series of
glycerine-based PEGs activated by a chemistry chosen by those
skilled in the art, any of the SUNBRITE activated PEGs (including
but not limited to carboxyl-PEGs, p-NP-PEGs, Tresyl-PEGs, aldehyde
PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs, maleimido-PEGs,
hydroxyl-PEG-amine, amino-PEG-COOH, hydroxyl-PEG-aldehyde,
carboxylic anhydride type-PEG, functionalized PEG-phospholipid, and
other similar and/or suitable reactive PEGs as selected by those
skilled in the art for their particular application and usage.
[0053] The novel peptides of the invention can also contain two PEG
moieties that are covalently attached via a carbamate or an amide
linkage to a spacer moiety, wherein the spacer moiety is covalently
bonded to the tertiary amide linker of the peptide dimer. Each of
the two PEG moieties used in such embodiments of the present
invention may be linear and may be linked together at a single
point of attachment. Each PEG moiety preferably has a molecular
weight of about 10 kilodaltons (10K) to about 60K (the term "about"
indicating that in preparations of PEG, some molecules will weigh
more, some less, than the stated molecular weight). Linear PEG
moieties are particularly preferred. More preferably, each of the
two PEG moieties has a molecular weight of about 20K to about 40K,
and still more preferably between about 20K and about 40K. Still
more preferably, each of the two PEG moieties has a molecular
weight of about 20K. One skilled in the art will be able to select
the desired polymer size based on such considerations as the
desired dosage; circulation time; resistance to proteolysis;
effects, if any, on biological activity; ease in handling; degree
or lack of antigenicity; and other known effects of PEG on a
therapeutic peptide.
[0054] The present invention also comprises peptide agonists that
are homodimers of peptide monomers having the amino acid sequence
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG) (SEQ ID NO: 3), where each amino
acid is indicated by standard one letter abbreviation, "(AcG)" is
N-acetylglycine, "(1-nal)" is 1-naphthylalanine, and "(MeG)" is
N-methylglycine, also known as sarcosine. Each peptide monomer of
the peptide dimer contains an intramolecular disulfide bond between
the cysteine residues of the monomer. Such monomers may be
represented schematically as follows:
##STR00016##
[0055] These monomeric peptides are dimerized to provide peptide
dimers of enhanced EPO-R agonist activity. The linker (L.sub.K)
moiety is a lysine residue, which bridges the C-termini of two
peptide monomers, by simultaneous attachment to the C-terminal
amino acid of each monomer. One peptide monomer is attached at its
C-terminus to the lysine's .epsilon.-amino group and the second
peptide monomer is attached at its C-terminus to the lysine's
.alpha.-amino group. For example, the dimer may be illustrated
structurally as shown in Formula I, and summarized as shown in
Formula II:
##STR00017##
In Formula I and Formula II, N.sup.2 represents the nitrogen atom
of lysine's .epsilon.-amino group and N.sup.1 represents the
nitrogen atom of lysine's .alpha.-amino group.
[0056] The peptide dimers of the invention further comprise a
spacer moiety of the following structure:
--N.sup.1H--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2---
N.sup.2H--
At one end, N.sup.1 of the spacer is attached via an amide linkage
to a carbonyl carbon of the lysine linker. At the opposite end,
N.sup.2 of the spacer is attached via a carbamate linkage or an
amide linkage to an activated polyethylene glycol (PEG) moiety,
where the PEG has a molecular weight of about 10,000 to about
60,000 Daltons (the term "about" indicating that in preparations of
PEG, some molecules will weigh more, some less, than the stated
molecular weight). More preferably, the PEG has a molecular weight
of about 20,000 to 40,000 Daltons.
[0057] Thus, the novel peptides of the invention also contain a PEG
moiety, which is covalently attached to the peptide dimer. PEG is a
water soluble polymer that is pharmaceutically acceptable. PEG for
use in the present invention may be linear, unbranched PEG having a
molecular weight of about 20 kilodaltons (20K) to about 60K (the
term "about" indicating that in preparations of PEG, some molecules
will weigh more, some less, than the stated molecular weight). Most
preferably, the PEG has a molecular weight of about 20K to about
40K, and still more preferably a molecular weight of about 30K to
about 40K. One skilled in the art will be able to select the
desired polymer size based on such considerations as the desired
dosage; circulation time; resistance to proteolysis; effects, if
any, on biological activity; ease in handling; degree or lack of
antigenicity; and other known effects of PEG on a therapeutic
peptide.
[0058] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N.sup.1
of the linker is attached via a carbamate linkage to an activated
polyethylene glycol (PEG) moiety, the novel peptide compounds of
the invention may be represented as follows:
##STR00018##
[0059] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N.sup.1
of the linker is attached via an amide linkage to an activated
polyethylene glycol (PEG) moiety, the novel peptide compounds of
the invention may be represented as follows:
##STR00019##
[0060] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
N.sup.1 of the linker is attached via a carbamate linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00020##
[0061] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
N.sup.1 of the linker is attached via an amide linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00021##
[0062] Preferred peptide dimers of the present invention include,
but are not limited to:
##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
##STR00027##
[0063] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both
N.sup.1 and N.sup.2 of the spacer are covalently attached via a
carbamate linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00028##
[0064] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both
N.sup.1 and N.sup.2 of the spacer are covalently attached via an
amide linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00029##
[0065] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
both N.sup.1 and N.sup.2 of the spacer are covalently attached via
a carbamate linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00030##
[0066] Where each monomer of the homodimer has the amino acid
sequence, (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and
both N.sup.1 and N.sup.2 of the spacer are covalently attached via
an amide linkage to an activated PEG moiety, the novel peptide
compounds of the invention may be represented as follows:
##STR00031##
[0067] Preferred peptide dimers of the present invention include,
but are not limited to:
##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##
##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041##
[0068] Where the spacer is attached via a carbamate linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention (SEQ ID NO: 3) may be represented as
follows:
##STR00042##
[0069] Where the spacer is attached via an amide linkage to an
activated polyethylene glycol (PEG) moiety, the novel peptide
compounds of the invention (SEQ ID NO: 3) may be represented as
follows:
##STR00043##
[0070] This dimeric structure can be written [Ac-peptide,
disulfide].sub.2Lys-spacer-PEG.sub.20-40K to denote an N-terminally
acetylated peptide bound to both the .alpha. and .epsilon. amino
groups of lysine with each peptide containing an intramolecular
disulfide loop and a spacer molecule forming a covalent linkage
between the C-terminus of lysine and a PEG moiety, where the PEG
has a molecular weight of about 20,000 to about 40,000 Daltons.
[0071] Preferred peptide dimers of the present invention include,
but are not limited to:
##STR00044## ##STR00045##
[0072] Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids, unnatural amino acids such as
a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid,
and other unconventional amino acids may also be suitable
components for compounds of the present invention. Examples of
unconventional amino acids include, but are not limited to:
.beta.-alanine, 3-pyridylalanine, 4-hydroxyproline,
O-phosphoserine, N-methylglycine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
nor-leucine, and other similar amino acids and imino acids. Other
modifications are also possible, including modification of the
amino terminus, modification of the carboxy terminus, replacement
of one or more of the naturally occurring genetically encoded amino
acids with an unconventional amino acid, modification of the side
chain of one or more amino acid residues, peptide phosphorylation,
and the like.
[0073] The peptide sequences of the present invention and be
present alone or in conjunction with N-terminal and/or C-terminal
extensions of the peptide chain. Such extensions may be naturally
encoded peptide sequences optionally with or substantially without
non-naturally occurring sequences; the extensions may include any
additions, deletions, point mutations, or other sequence
modifications or combinations as desired by those skilled in the
art. For example and not limitation, naturally-occurring sequences
may be full-length or partial length and may include amino acid
substitutions to provide a site for attachment of carbohydrate,
PEG, other polymer, or the like via side chain conjugation. In a
variation, the amino acid substitution results in humanization of a
sequence to make in compatible with the human immune system. Fusion
proteins of all types are provided, including immunoglobulin
sequences adjacent to or in near proximity to the EPO-R activating
sequences of the present invention with or without a
non-immunoglobulin spacer sequence. One type of embodiment is an
immunoglobulin chain having the EPO-R activating sequence in place
of the variable (V) region of the heavy and/or light chain.
Preparation of the Peptide Compounds of the Invention
Peptide Synthesis
[0074] The peptides of the invention may be prepared by classical
methods known in the art. These standard methods include exclusive
solid phase synthesis, partial solid phase synthesis methods,
fragment condensation, classical solution synthesis, and
recombinant DNA technology [See, e.g., Merrifield J. Am. Chem. Soc.
1963 85:2149].
[0075] In one embodiment, the peptide monomers of a peptide dimer
are synthesized individually and dimerized subsequent to
synthesis.
[0076] In another embodiment, the peptide monomers of a dimer are
linked via their C-termini by a branched tertiary amide linker
L.sub.K moiety having two functional groups capable of serving as
initiation sites for peptide synthesis and a third functional group
(e.g., a carboxyl group or an amino group) that enables binding to
another molecular moiety (e.g., as may be present on the surface of
a solid support). In this case, the two peptide monomers may be
synthesized directly onto two reactive nitrogen groups of the
linker L.sub.K moiety in a variation of the solid phase synthesis
technique. Such synthesis may be sequential or simultaneous.
[0077] In another embodiment, the two peptide monomers may be
synthesized directly onto two reactive nitrogen groups of the
linker L.sub.K moiety in a variation of the solid phase synthesis
technique. Such synthesis may be sequential or simultaneous. In
this embodiment, a lysine linker (L.sub.K) moiety having two amino
groups capable of serving as initiation sites for peptide synthesis
and a third functional group (e.g., the carboxyl group of a lysine;
or the amino group of a lysine amide, a lysine residue wherein the
carboxyl group has been converted to an amide moiety --CONH.sub.2)
that enables binding to another molecular moiety (e.g., as may be
present on the surface of a solid support) is used.
[0078] Where sequential synthesis of the peptide chains of a dimer
onto a linker is to be performed, two amine functional groups on
the linker molecule are protected with two different orthogonally
removable amine protecting groups. The protected linker is coupled
to a solid support via the linker's third functional group. The
first amine protecting group is removed, and the first peptide of
the dimer is synthesized on the first deprotected amine moiety.
Then the second amine protecting group is removed, and the second
peptide of the dimer is synthesized on the second deprotected amine
moiety. For example, the first amino moiety of the linker may be
protected with Alloc, and the second with Fmoc. In this case, the
Fmoc group (but not the Alloc group) may be removed by treatment
with a mild base [e.g., 20% piperidine in dimethyl formamide
(DMF)], and the first peptide chain synthesized. Thereafter the
Alloc group may be removed with a suitable reagent [e.g.,
Pd(PPh.sub.3)/4-methyl morpholine and chloroform], and the second
peptide chain synthesized. Note that where different
thiol-protecting groups for cysteine are to be used to control
disulfide bond formation (as discussed below) this technique must
be used even where the final amino acid sequences of the peptide
chains of a dimer are identical.
[0079] Where simultaneous synthesis of the peptide chains of a
dimer onto a linker is to be performed, two amine functional groups
of the linker molecule are protected with the same removable amine
protecting group. The protected linker is coupled to a solid
support via the linker's third functional group. In this case the
two protected functional groups of the linker molecule are
simultaneously deprotected, and the two peptide chains
simultaneously synthesized on the deprotected amines. Note that
using this technique, the sequences of the peptide chains of the
dimer will be identical, and the thiol-protecting groups for the
cysteine residues are all the same.
[0080] A preferred method for peptide synthesis is solid phase
synthesis. Solid phase peptide synthesis procedures are well-known
in the art [see, e.g., Stewart Solid Phase Peptide Syntheses
(Freeman and Co.: San Francisco) 1969; 2002/2003 General Catalog
from Novabiochem Corp, San Diego, USA; Goodman Synthesis of
Peptides and Peptidomimetics (Houben-Weyl, Stuttgart) 2002]. In
solid phase synthesis, synthesis is typically commenced from the
C-terminal end of the peptide using an .alpha.-amino protected
resin. A suitable starting material can be prepared, for instance,
by attaching the required .alpha.-amino acid to a chloromethylated
resin, a hydroxymethyl resin, a polystyrene resin, a
benzhydrylamine resin, or the like. One such chloromethylated resin
is sold under the trade name BIO-BEADS SX-1 by Bio Rad Laboratories
(Richmond, Calif.). The preparation of the hydroxymethyl resin has
been described [Bodonszky, et al. (1966) Chem. Ind. London
38:1597]. The benzhydrylamine (BHA) resin has been described
[Pietta and Marshall (1970) Chem. Commun. 650], and the
hydrochloride form is commercially available from Beckman
Instruments, Inc. (Palo Alto, Calif.). For example, an
.alpha.-amino protected amino acid may be coupled to a
chloromethylated resin with the aid of a cesium bicarbonate
catalyst, according to the method described by Gisin (1973) Helv.
Chim. Acta 56:1467.
[0081] After initial coupling, the .alpha.-amino protecting group
is removed, for example, using trifluoroacetic acid (TFA) or
hydrochloric acid (HCl) solutions in organic solvents at room
temperature. Thereafter, .alpha.-amino protected amino acids are
successively coupled to a growing support-bound peptide chain. The
.alpha.-amino protecting groups are those known to be useful in the
art of stepwise synthesis of peptides, including: acyl-type
protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic
urethane-type protecting groups [e.g., benzyloxycarboyl (Cbz) and
substituted Cbz], aliphatic urethane protecting groups [e.g.,
t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,
cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g.,
benzyl, triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc),
allyloxycarbonyl (Alloc), and
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde).
[0082] The side chain protecting groups (typically ethers, esters,
trityl, PMC, and the like) remain intact during coupling and are
not split off during the deprotection of the amino-terminus
protecting group or during coupling. The side chain protecting
group must be removable upon the completion of the synthesis of the
final peptide and under reaction conditions that will not alter the
target peptide. The side chain protecting groups for Tyr include
tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z--Br--Cbz, and
2,5-dichlorobenzyl. The side chain protecting groups for Asp
include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl.
The side chain protecting groups for Thr and Ser include acetyl,
benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and
Cbz. The side chain protecting groups for Arg include nitro, Tosyl
(Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts),
2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),
4-methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), or Boc. The side
chain protecting groups for Lys include Cbz,
2-chlorobenzyloxycarbonyl (2-Cl--Cbz), 2-bromobenzyloxycarbonyl
(2-Br--Cbz), Tos, or Boc.
[0083] After removal of the .alpha.-amino protecting group, the
remaining protected amino acids are coupled stepwise in the desired
order. Each protected amino acid is generally reacted in about a
3-fold excess using an appropriate carboxyl group activator such as
2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium
hexafluorophosphate (HBTU) or dicyclohexylcarbodiimide (DCC) in
solution, for example, in methylene chloride (CH.sub.2Cl.sub.2),
N-methylpyrrolidone, dimethyl formamide (DMF), or mixtures
thereof.
[0084] After the desired amino acid sequence has been completed,
the desired peptide is decoupled from the resin support by
treatment with a reagent, such as trifluoroacetic acid (TFA) or
hydrogen fluoride (HF), which not only cleaves the peptide from the
resin, but also cleaves all remaining side chain protecting groups.
When a chloromethylated resin is used, hydrogen fluoride treatment
results in the formation of the free peptide acids. When the
benzhydrylamine resin is used, hydrogen fluoride treatment results
directly in the free peptide amide. Alternatively, when the
chloromethylated resin is employed, the side chain protected
peptide can be decoupled by treatment of the peptide resin with
ammonia to give the desired side chain protected amide or with an
alkylamine to give a side chain protected alkylamide or
dialkylamide. Side chain protection is then removed in the usual
fashion by treatment with hydrogen fluoride to give the free
amides, alkylamides, or dialkylamides.
[0085] In preparing the esters of the invention, the resins used to
prepare the peptide acids are employed, and the side chain
protected peptide is cleaved with base and the appropriate alcohol
(e.g., methanol). Side chain protecting groups are then removed in
the usual fashion by treatment with hydrogen fluoride to obtain the
desired ester.
[0086] These procedures can also be used to synthesize peptides in
which amino acids other than the 20 naturally occurring,
genetically encoded amino acids are substituted at one, two, or
more positions of any of the compounds of the invention. Synthetic
amino acids that can be substituted into the peptides of the
present invention include, but are not limited to, N-methyl,
L-hydroxypropyl, L-3,4-dihydrooxyphenylalanyl, .delta. amino acids
such as L-.delta.-hydroxylysyl and D-.delta.-methylalanyl,
L-.alpha.-methylalanyl, .beta. amino acids, and isoquinolyl.
D-amino acids and non-naturally occurring synthetic amino acids can
also be incorporated into the peptides of the present
invention.
Peptide Modifications
[0087] One can also modify the amino and/or carboxy termini of the
peptide compounds of the invention to produce other compounds of
the invention. For example, the amino terminus may be acetylated
with acetic acid or a halogenated derivative thereof such as
.alpha.-chloroacetic acid, .alpha.-bromoacetic acid, or
.alpha.-iodoacetic acid).
[0088] One can replace the naturally occurring side chains of the
20 genetically encoded amino acids (or the stereoisomeric D amino
acids) with other side chains, for instance with groups such as
alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide,
amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,
carboxy and the lower ester derivatives thereof, and with 4-, 5-,
6-, to 7-membered heterocyclic. In particular, proline analogues in
which the ring size of the praline residue is changed from 5
members to 4, 6, or 7 members can be employed. Cyclic groups can be
saturated or unsaturated, and if unsaturated, can be aromatic or
non-aromatic. Heterocyclic groups preferably contain one or more
nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such
groups include the furazanyl, furyl, imidazolidinyl, imidazolyl,
imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.
morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl
(e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,
pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,
thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,
thiomorpholino), and triazolyl. These heterocyclic groups can be
substituted or unsubstituted. Where a group is substituted, the
substituent can be alkyl, alkoxy, halogen, oxygen, or substituted
or unsubstituted phenyl.
[0089] One can also readily modify peptides by phosphorylation, and
other methods [e.g., as described in Hruby, et al. (1990) Biochem
J. 268:249-262].
[0090] The peptide compounds of the invention also serve as
structural models for non-peptidic compounds with similar
biological activity. Those of skill in the art recognize that a
variety of techniques are available for constructing compounds with
the same or similar desired biological activity as the lead peptide
compound, but with more favorable activity than the lead with
respect to solubility, stability, and susceptibility to hydrolysis
and proteolysis [See, Morgan and Gainor (1989) Ann. Rep. Med. Chem.
24:243-252]. These techniques include replacing the peptide
backbone with a backbone composed of phosphonates, amidates,
carbamates, sulfonamides, secondary amines, and N-methylamino
acids.
Formation of Disulfide Bonds
[0091] The compounds of the present invention contain two
intramolecular disulfide bonds. Such disulfide bonds may be formed
by oxidation of the cysteine residues of each peptide monomer.
[0092] In one embodiment, the control of cysteine bond formation is
exercised by choosing an oxidizing agent of the type and
concentration effective to optimize formation of the desired
isomer. For example, oxidation of a peptide dimer to form two
intramolecular disulfide bonds (one on each peptide chain) is
preferentially achieved (over formation of intermolecular disulfide
bonds) when the oxidizing agent is DMSO or iodine (I.sub.2). In
other embodiments, the formation of cysteine bonds is controlled by
the selective use of thiol-protecting groups during peptide
synthesis. For example, where a dimer with two intramolecular
disulfide bonds is desired, the first monomer peptide chain is
synthesized with the two cysteine residues of the core sequence
protected with a first thiol protecting group [e.g., trityl(Trt),
allyloxycarbonyl (Alloc), and
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) or the
like], then the second monomer peptide is synthesized the two
cysteine residues of the core sequence protected with a second
thiol protecting group different from the first thiol protecting
group [e.g., acetamidomethyl (Acm), t-butyl (tBu), or the like].
Thereafter, the first thiol protecting groups are removed effecting
bisulfide cyclization of the first monomer, and then the second
thiol protecting groups are removed effecting bisulfide cyclization
of the second monomer.
[0093] Other embodiments of this invention provide for analogues of
these disulfide derivatives in which one of the sulfurs has been
replaced by a CH.sub.2 group or other isotere for sulfur. These
analogues can be prepared from the compounds of the present
invention, wherein each peptide monomer contains at least one C or
homocysteine residue and an .alpha.-amino-.gamma.-butyric acid in
place of the second C residue, via an intramolecular or
intermolecular displacement, using methods known in the art [See,
e.g., Barker, et al. (1992) J. Med. Chem. 35:2040-2048 and Or, et
al. (1991) J. Org. Chem. 56:3146-3149]. One of skill in the art
will readily appreciate that this displacement can also occur using
other homologs of .alpha.-amino-.gamma.-butyric acid and
homocysteine.
[0094] In addition to the foregoing cyclization strategies, other
non-disulfide peptide cyclization strategies can be employed. Such
alternative cyclization strategies include, for example,
amide-cyclization strategies as well as those involving the
formation of thio-ether bonds. Thus, the compounds of the present
invention can exist in a cyclized form with either an
intramolecular amide bond or an intramolecular thio-ether bond. For
example, a peptide may be synthesized wherein one cysteine of the
core sequence is replaced with lysine and the second cysteine is
replaced with glutamic acid. Thereafter a cyclic monomer may be
formed through an amide bond between the side chains of these two
residues. Alternatively, a peptide may be synthesized wherein one
cysteine of the core sequence is replaced with lysine (or serine).
A cyclic monomer may then be formed through a thio-ether linkage
between the side chains of the lysine (or serine) residue and the
second cysteine residue of the core sequence. As such, in addition
to disulfide cyclization strategies, amide-cyclization strategies
and thio-ether cyclization strategies can both be readily used to
cyclize the compounds of the present invention. Alternatively, the
amino-terminus of the peptide can be capped with an
.alpha.-substituted acetic acid, wherein the .alpha.-substituent is
a leaving group, such as an .alpha.-haloacetic acid, for example,
.alpha.-chloroacetic acid, .alpha.-bromoacetic acid, or
.alpha.-iodoacetic acid.
Addition of Branched Tertiary Amide Linker
[0095] The peptide monomers may be dimerized by a branched tertiary
amide linker moiety. In one embodiment, the linker is incorporated
into the peptide during peptide synthesis. For example, where a
linker L.sub.K moiety contains two functional groups capable of
serving as initiation sites for peptide synthesis and one or more
other functional groups (e.g., a carboxyl group or an amino group)
that enables binding to one or more other molecular moieties, the
linker may be conjugated to a solid support. Thereafter, two
peptide monomers may be synthesized directly onto the two reactive
nitrogen groups of the linker L.sub.K moiety in a variation of the
solid phase synthesis technique.
[0096] In alternate embodiments, the linker may be conjugated to
the two peptide monomers of a peptide dimer after peptide
synthesis. Such conjugation may be achieved by methods well
established in the art. In one embodiment, the linker contains two
functional groups suitable for attachment to the target functional
groups of the synthesized peptide monomers. For example, a linker
containing two carboxyl groups, either preactivated or in the
presence of a suitable coupling reagent, may be reacted with the
target lysine side chain amine groups of each of two peptide
monomers.
[0097] For example, the peptide monomers may be chemically coupled
to the tertiary amide linker,
A*--C.sup.1O--CH.sub.2--X--CH.sub.2--C.sup.2O--B*
where: X is NCO--(CH.sub.2).sub.2--NH--Y and Y is a suitable
protecting group, such as a t-butyloxycarbonyl (Boc) protecting
group; A* is a suitable functional group, such as N-oxy
succinimide, used to conjugate C.sup.1 of the linker to the
.epsilon.-amino group of the C-terminal lysine residue of the first
peptide monomer; and B* is a suitable functional group, such as
N-oxy succinimide, used to conjugate C.sup.2 of the linker to the
.epsilon.-amino group of the C-terminal lysine residue of the
second peptide monomer.
[0098] Additionally, for example, the peptide monomers may be
chemically coupled to the tertiary amide linker,
A*--C.sup.1O--CH.sub.2--X--CH.sub.2--C.sup.2O--B*
where: X is NCO--(CH.sub.2).sub.2--NH--C.sup.3O--; A* is a suitable
functional group, such as N-oxy succinimide, used to conjugate
C.sup.1 of the linker to the .epsilon.-amino group of the
C-terminal lysine residue of the first peptide monomer; and B* is a
suitable functional group, such as N-oxy succinimide, used to
conjugate C.sup.2 of the linker to the .epsilon.-amino group of the
C-terminal lysine residue of the second peptide monomer; and the
tertiary amide linker is chemically bonded to the spacer
moiety,
Y--NH--(CH.sub.2).sub.4--C.sup.4H--NH--Y
where: C.sup.3 of X is covalently bonded to C.sup.4 of the spacer;
and Y is a suitable protecting group, such as a t-butyloxycarbonyl
(Boc) protecting group.
Addition of Lysine Linker
[0099] The peptide monomers may be dimerized by a lysine linker
L.sub.K moiety. In one embodiment, the lysine linker in
incorporated into the peptide during peptide synthesis. For
example, where a lysine linker L.sub.K moiety contains two
functional groups capable of serving as initiation sites for
peptide synthesis and a third functional group (e.g., a carboxyl
group or an amino group) that enables binding to another molecular
moiety, the linker may be conjugated to a solid support.
Thereafter, two peptide monomers may be synthesized directly onto
the two reactive nitrogen groups of the lysine linker L.sub.K
moiety in a variation of the solid phase synthesis technique.
[0100] In alternate embodiments where a peptide dimer is dimerized
by a lysine linker L.sub.K moiety, said linker may be conjugated to
the two peptide monomers of a peptide dimer after peptide
synthesis. Such conjugation may be achieved by methods well
established in the art. In one embodiment, the linker contains at
least two functional groups suitable for attachment to the target
functional groups of the synthesized peptide monomers. For example,
the lysine's two free amine groups may be reacted with the
C-terminal carboxyl groups of each of two peptide monomers.
Addition of Spacer
[0101] The peptide compounds of the invention further comprise a
spacer moiety. In one embodiment the spacer may be incorporated
into the peptide during peptide synthesis. For example, where a
spacer contains a free amino group and a second functional group
(e.g., a carboxyl group or an amino group) that enables binding to
another molecular moiety, the spacer may be conjugated to the solid
support.
[0102] In one embodiment, a spacer containing two functional groups
is first coupled to the solid support via a first functional group.
Next the lysine linker L.sub.K moiety having two functional groups
capable of serving as initiation sites for peptide synthesis and a
third functional group (e.g., a carboxyl group or an amino group)
that enables binding to another molecular moiety is conjugated to
the spacer via the spacer's second functional group and the
linker's third functional group. Thereafter, two peptide monomers
may be synthesized directly onto the two reactive nitrogen groups
of the linker L.sub.K moiety in a variation of the solid phase
synthesis technique. For example, a solid support coupled spacer
with a free amine group may be reacted with a lysine linker via the
linker's free carboxyl group.
[0103] In alternate embodiments the spacer may be conjugated to the
peptide dimer after peptide synthesis. Such conjugation may be
achieved by methods well established in the art. In one embodiment,
the linker contains at least one functional group suitable for
attachment to the target functional group of the synthesized
peptide. For example, a spacer with a free amine group may be
reacted with a peptide's C-terminal carboxyl group. In another
example, a linker with a free carboxyl group may be reacted with
the free amine group of a lysine amide.
Attachment of Polyethylene Glycol (PEG)
[0104] In recent years, water-soluble polymers, such as
polyethylene glycol (PEG), have been used for the covalent
modification of peptides of therapeutic and diagnostic importance.
Attachment of such polymers is thought to enhance biological
activity, prolong blood circulation time, reduce immunogenicity,
increase aqueous solubility, and enhance resistance to protease
digestion. For example, covalent attachment of PEG to therapeutic
polypeptides such as interleukins [Knauf, et al. (1988) J. Biol.
Chem. 263; 15064; Tsutsumi, et al. (1995) J. Controlled Release
33:447), interferons (Kita, et al. (1990) Drug Des. Delivery
6:157), catalase (Abuchowski, et al. (1977) J. Biol. Chem.
252:582), superoxide dismutase (Beauchamp, et al. (1983) Anal.
Biochem. 131:25), and adenosine deaminase (Chen, et al. (1981)
Biochim. Biophy. Acta 660:293), has been reported to extend their
half life in vivo, and/or reduce their immunogenicity and
antigenicity.
[0105] The peptide compounds of the invention may comprise a
polyethylene glycol (PEG) moiety, which is covalently attached to
the branched tertiary amide linker or the spacer of the peptide
dimer via a carbamate linkage or via an amide linkage. An example
of PEG used in the present invention is linear, unbranched PEG
having a molecular weight of about 20 kiloDaltons (20K) to about
40K (the term "about" indicating that in preparations of PEG, some
molecules will weigh more, some less, than the stated molecular
weight). Preferably, the PEG has a molecular weight of about 30K to
about 40K.
[0106] Another example of PEG used in the present invention is
linear PEG having a molecular weight of about 10K to about 60K (the
term "about" indicating that in preparations of PEG, some molecules
will weigh more, some less, than the stated molecular weight).
Preferably, the PEG has a molecular weight of about 20K to about
40K. More preferably, the PEG has a molecular weight of about
20K.
[0107] Examples of methods for covalent attachment of PEG
(PEGylation) are described below. These illustrative descriptions
are not intended to be limiting. One of ordinary skill in the art
will appreciate that a variety of methods for covalent attachment
of a broad range of PEG is well established in the art. As such,
peptide compounds to which PEG has been attached by any of a number
of attachment methods known in the art are encompassed by the
present invention.
[0108] For example, PEG may be covalently bound to the linker via a
reactive group to which an activated PEG molecule may be bound
(e.g., a free amino group or carboxyl group). PEG molecules may be
attached to amino groups using methoxylated PEG ("mPEG") having
different reactive moieties. Such polymers include
mPEG-succinimidyl succinate, mPEG-succinimidyl carbonate,
mPEG-imidate, mPEG-4-nitrophenyl carbonate, and mPEG-cyanuric
chloride. Similarly, PEG molecules may be attached to carboxyl
groups using methoxylated PEG with a free amine group
(mPEG-NH.sub.2).
[0109] In some embodiments, the linker or spacer contains a
terminal amino group (i.e., positioned at the terminus of the
spacer). This terminal amino group may be reacted with a suitably
activated PEG molecule, such as mPEG-para-nitrophenylcarbonate
(mPEG-NPC), to make a stable covalent carbamate bond.
Alternatively, this terminal amino group may be reacted with a
suitably activated PEG molecule, such as an mPEG-succinimidyl
butyrate (mPEG-SBA) or in PEG-succinimidyl propionate (mPEG-SPA)
containing a reactive N-hydroxyl-succinimide (NHS) group, to make a
stable covalent carbamate bond. In other embodiments, the linker
reactive group contains a carboxyl group capable of being activated
to form a covalent bond with an amine-containing PEG molecule under
suitable reaction conditions. Suitable PEG molecules include
mPEG-NH.sub.2 and suitable reaction conditions include
carbodiimide-mediated amide formation or the like.
EPO-R Agonist Activity Assays:
In Vitro Functional Assays
[0110] In vitro competitive binding assays quantitate the ability
of a test peptide to compete with EPO for binding to EPO-R. For
example (see, e.g., as described in U.S. Pat. No. 5,773,569), the
extracellular domain of the human EPO-R (EPO binding protein, EBP)
may be recombinantly produced in E. coli and the recombinant
protein coupled to a solid support, such as a microtitre dish or a
synthetic bead [e.g., Sulfolink beads from Pierce Chemical Co.
(Rockford, Ill.)]. Immobilized EBP is then incubated with labeled
recombinant EPO, or with labeled recombinant EPO and a test
peptide. Serial dilutions of test peptide are employed for such
experiments. Assay points with no added test peptide define total
EPO binding to EBP. For reactions containing test peptide, the
amount of bound EPO is quantitated and expressed as a percentage of
the control (total=100%) binding. These values are plotted versus
peptide concentration. The IC50 value is defined as the
concentration of test peptide which reduces the binding of EPO to
EBP by 50% (i.e., 50% inhibition of EPO binding).
[0111] A different in vitro competitive binding assay measures the
light signal generated as a function of the proximity of two beads:
an EPO-conjugated bead and an EPO-R-conjugated bead. Bead proximity
is generated by the binding of EPO to EPO-R. A test peptide that
competes with EPO for binding to EPO-R will prevent this binding,
causing a decrease in light emission. The concentration of test
peptide that results in a 50% decrease in light emission is defined
as the 1050 value.
[0112] The peptides of the present invention compete very
efficiently with EPO for binding to the EPO-R. This enhanced
function is represented by their ability to inhibit the binding of
EPO at substantially lower concentrations of peptide (i.e., they
have very low IC50 values).
[0113] The biological activity and potency of monomeric and dimeric
peptide EPO-R agonists of the invention, which bind specifically to
the EPO-receptor, may be measured using in vitro cell-based
functional assays.
[0114] One assay is based upon a murine pre-B-cell line expressing
human EPO-R and further transfected with a fos promoter-driven
luciferase reporter gene construct. Upon exposure to EPO or another
EPO-R agonist, such cells respond by synthesizing luciferase.
Luciferase causes the emission of light upon addition of its
substrate luciferin. Thus, the level of EPO-R activation in such
cells may be quantitated via measurement of luciferase activity.
The activity of a test peptide is measured by adding serial
dilutions of the test peptide to the cells, which are then
incubated for 4 hours. After incubation, luciferin substrate is
added to the cells, and light emission is measured. The
concentration of test peptide that results in a half-maximal
emission of light is recorded as the EC50.
[0115] The peptides of the present invention show dramatically
enhanced ability to promote EPO-R signaling-dependent luciferase
expression in this assay. This enhanced function is represented by
their ability to yield half of the maximal luciferase activity at
substantially lower concentrations of peptide (i.e., they have very
low EC50 values). This assay is a preferred method for estimating
the potency and activity of an EPO-R agonist peptide of the
invention.
[0116] Another assay may be performed using FDC-P1/ER cells
[Dexter, et al. (1980) J. Exp. Med. 152:1036-1047], a well
characterized nontransformed murine bone marrow derived cell line
into which EPO-R has been stably transfected. These cells exhibit
EPO-dependent proliferation.
[0117] In one such assay, the cells are grown to half stationary
density in the presence of the necessary growth factors (see, e.g.,
as described in U.S. Pat. No. 5,773,569). The cells are then washed
in PBS and starved for 16-24 hours in whole media without the
growth factors. After determining the viability of the cells (e.g.,
by trypan blue staining), stock solutions (in whole media without
the growth factors) are made to give about 10.sup.5 cells per 50
.mu.L. Serial dilutions of the peptide EPO-R agonist compounds
(typically the free, solution phase peptide as opposed to a
phage-bound or other bound or immobilized peptide) to be tested are
made in 96-well tissue culture plates for a final volume of 50
.mu.L per well. Cells (50 .mu.L) are added to each well and the
cells are incubated 24-48 hours, at which point the negative
controls should die or be quiescent. Cell proliferation is then
measured by techniques known in the art, such as an MTT assay which
measures H.sup.3-thymidine incorporation as an indication of cell
proliferation [see, Mosmann (1983) J. Immunol. Methods 65:55-63].
Peptides are evaluated on both the EPO-R-expressing cell line and a
parental non-expressing cell line. The concentration of test
peptide necessary to yield one half of the maximal cell
proliferation is recorded as the EC50.
[0118] The peptides of the present invention show dramatically
enhanced ability to promote EPO-dependent cell growth in this
assay. This enhanced function is represented by their ability to
yield half of the maximal cell proliferation stimulation activity
at substantially lower concentrations of peptide (i.e., they have
very low EC50 values). This assay is a preferred method for
estimating the potency and activity of an EPO-R agonist peptide of
the invention.
[0119] In another assay, the cells are grown to stationary phase in
EPO-supplemented medium, collected, and then cultured for an
additional 18 hr in medium without EPO. The cells are divided into
three groups of equal cell density: one group with no added factor
(negative control), a group with EPO (positive control), and an
experimental group with the test peptide. The cultured cells are
then collected at various time points, fixed, and stained with a
DNA-binding fluorescent dye (e.g., propidium iodide or Hoechst dye,
both available from Sigma). Fluorescence is then measured, for
example, using a FACS Scan Flow cytometer. The percentage of cells
in each phase of the cell cycle may then be determined, for
example, using the SOBR model of CelIFIT software (Becton
Dickinson). Cells treated with EPO or an active peptide will show a
greater proportion of cells in S phase (as determined by increased
fluorescence as an indicator of increased DNA content) relative to
the negative control group.
[0120] Similar assays may be performed using FDCP-1 [see, e.g.,
Dexter et al. (1980) J. Exp. Med. 152:1036-1047] or TF-1 [Kitamura,
et al. (1989) Blood 73:375-380] cell lines. FDCP-1 is a growth
factor dependent murine multi-potential primitive hematopoietic
progenitor cell line that can proliferate, but not differentiate,
when supplemented with WEHI-3-conditioned media (a medium that
contains IL-3, ATCC number TIB-68). For such experiments, the
FDCP-1 cell line is transfected with the human or murine EPO-R to
produce FDCP-1-hEPO-R or FDCP-1-mEPO-R cell lines, respectively,
that can proliferate, but not differentiate, in the presence of
EPO. TF-1, an EPO-dependent cell line, may also be used to measure
the effects of peptide EPO-R agonists on cellular
proliferation.
[0121] In yet another assay, the procedure set forth in Krystal
(1983) Exp. Hematol 11:649-660 for a microassay based on
H.sup.3-thymidine incorporation into spleen cells may be employed
to ascertain the ability of the compounds of the present invention
to serve as EPO agonists. In brief, B6C3F.sub.1 mice are injected
daily for two days with phenylhydrazine (60 mg/kg). On the third
day, spleen cells are removed and their ability to proliferate over
a 24 hour period ascertained using an MTT assay.
[0122] The binding of EPO to EPO-R in an erythropoietin-responsive
cell line induces tyrosine phosphorylation of both the receptor and
numerous intracellular proteins, including Shc, vav and JAK2
kinase. Therefore, another in vitro assay measures the ability of
peptides of the invention to induce tyrosine phosphorylation of
EPO-R and downstream intracellular signal transducer proteins.
Active peptides, as identified by binding and proliferation assays
described above, elicit a phosphorylation pattern nearly identical
to that of EPO in erythropoietin-responsive cells. For this assay,
FDC-P1/ER cells [Dexter, et al. (1980) J Exp Med 152:1036-47] are
maintained in EPO-supplemented medium and grown to stationary
phase. These cells are then cultured in medium without EPO for 24
hr. A defined number of such cells is then incubated with a test
peptide for approximately 10 min at 37.degree. C. A control sample
of cells with EPO is also run with each assay. The treated cells
are then collected by centrifugation, resuspended in SDS lysis
buffer, and subjected to SDS polyacrylamide gel electrophoresis.
The electrophoresed proteins in the gel are transferred to
nitrocellulose, and the phosphotyrosine containing proteins on the
blot visualized by standard immunological techniques. For example,
the blot may be probed with an anti-phosphotyrosine antibody (e.g.,
mouse anti-phosphotyrosine IgG from Upstate Biotechnology, Inc.),
washed, and then probed with a secondary antibody [e.g., peroxidase
labeled goat anti-mouse IgG from Kirkegaard & Perry
Laboratories, Inc. (Washington, D.C.)]. Thereafter,
phosphotyrosine-containing proteins may be visualized by standard
techniques including colorimetric, chemiluminescent, or fluorescent
assays. For example, a chemiluminescent assay may be performed
using the ECL Western Blotting System from Amersham.
[0123] Another cell-based in vitro assay that may be used to assess
the activity of the peptides of the present invention is a colony
assay, using murine bone marrow or human peripheral blood cells.
Murine bone marrow may be obtained from the femurs of mice, while a
sample of human peripheral blood may be obtained from a healthy
donor. In the case of peripheral blood, mononuclear cells are first
isolated from the blood, for example, by centrifugation through a
Ficoll-Hypaque gradient [Stem Cell Technologies, Inc. (Vancouver,
Canada)]. For this assay a nucleated cell count is performed to
establish the number and concentration of nucleated cells in the
original sample. A defined number of cells is plated on methyl
cellulose as per manufacturer's instructions [Stem Cell
Technologies, Inc. (Vancouver, Canada)]. An experimental group is
treated with a test peptide, a positive control group is treated
with EPO, and a negative control group receives no treatment. The
number of growing colonies for each group is then scored after
defined periods of incubation, generally 10 days and 18 days. An
active peptide will promote colony formation.
[0124] Other in vitro biological assays that can be used to
demonstrate the activity of the compounds of the present invention
are disclosed in Greenberger, et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2931-2935 (EPO-dependent hematopoietic progenitor cell
line); Quelle and Wojchowski (1991) J. Biol. Chem. 266:609-614
(protein tyrosine phosphorylation in B6SUt.EP cells);
Dusanter-Fourt, et al. (1992) J. Biol. Chem. 287:10670-10678
(tyrosine phosphorylation of EPO-receptor in human EPO-responsive
cells); Quelle, et al. (1992) J. Biol. Chem. 267:17055-17060
(tyrosine phosphorylation of a cytosolic protein, pp 100, in FDC-ER
cells); Worthington, et al. (1987) Exp. Hematol. 15:85-92
(colorimetric assay for hemoglobin); Kaiho and Miuno (1985) Anal.
Biochem. 149:117-120 (detection of hemoglobin with
2,7-diaminofluorene); Patel, et al. (1992) J. Biol. Chem.
267:21300-21302 (expression of c-myb); Witthuhn, et al. (1993) Cell
74:227-236 (association and tyrosine phosphorylation of JAK2);
Leonard, et al. (1993) Blood 82:1071-1079 (expression of GATA
transcription factors); and Ando, et al. (1993) Proc. Natl. Acad.
Sci. USA 90:9571-9575 (regulation of G.sub.1 transition by cycling
D2 and D3).
[0125] An instrument designed by Molecular Devices Corp., known as
a microphysiometer, has been reported to be successfully used for
measurement of the effect of agonists and antagonists on various
receptors. The basis for this apparatus is the measurement of the
alterations in the acidification rate of the extracellular media in
response to receptor activation.
In Vivo Functional Assays
[0126] One in vivo functional assay that may be used to assess the
potency of a test peptide is the polycythemic exhypoxic mouse
bioassay. For this assay, mice are subjected to an alternating
conditioning cycle for several days. In this cycle, the mice
alternate between periods of hypobaric conditions and ambient
pressure conditions. Thereafter, the mice are maintained at ambient
pressure for 2-3 days prior to administration of test samples. Test
peptide samples, or EPO standard in the case positive control mice,
are injected subcutaneously into the conditioned mice. Radiolabeled
iron (e.g., .sup.59Fe) is administered 2 days later, and blood
samples taken two days after administration of radiolabeled iron.
Hematocrits and radioactivity measurements are then determined for
each blood sample by standard techniques. Blood samples from mice
injected with active test peptides will show greater radioactivity
(due to binding of Fe.sup.59 by erythrocyte hemoglobin) than mice
that did not receive test peptides or EPO.
[0127] Another in viva functional assay that may be used to assess
the potency of a test peptide is the reticulocyte assay. For this
assay, normal untreated mice are subcutaneously injected on three
consecutive days with either EPO or test peptide. On the third day,
the mice are also intraperitoneally injected with iron dextran. At
day five, blood samples are collected from the mice. The percent
(%) of reticulocytes in the blood is determined by thiazole orange
staining and flow cytometer analysis (retic-count program). In
addition, hematocrits are manually determined. The percent of
corrected reticulocytes is determined using the following
formula:
% RETIC.sub.CORRECTED=%
RETIC.sub.OBSERVED.times.(Hematocrit.sub.INDIVIDUAL/Hematocrit.sub.NORMAL-
)
Active test compounds will show an increased % RETIC.sub.CORRECTED
level relative to mice that did not receive test peptides or
EPO.
Use of EPO-R Agonist Peptides of the Invention
[0128] The peptide compounds of the invention are useful in vitro
as tools for understanding the biological role of EPO, including
the evaluation of the many factors thought to influence, and be
influenced by, the production of EPO and the binding of EPO to the
EPO-R (e.g., the mechanism of EPO/EPO-R signal
transduction/receptor activation). The present peptides are also
useful in the development of other compounds that bind to the
EPO-R, because the present compounds provide important
structure-activity-relationship information that facilitates that
development.
[0129] Moreover, based on their ability to bind to EPO-R, the
peptides of the present invention can be used as reagents for
detecting EPO-R on living cells; fixed cells; in biological fluids;
in tissue homogenates; in purified, natural biological materials;
etc. For example, by labeling such peptides, one can identify cells
having EPO-R on their surfaces. In addition, based on their ability
to bind EPO-R, the peptides of the present invention can be used in
in situ staining, FACS (fluorescence-activated cell sorting)
analysis, Western blotting, ELISA (enzyme-linked immunosorbent
assay), etc. In addition, based on their ability to bind to EPO-R,
the peptides of the present invention can be used in receptor
purification or in purifying cells expressing EPO-R on the cell
surface (or inside permeabilized cells).
[0130] The peptides of the invention can also be utilized as
commercial reagents for various medical research and diagnostic
purposes. Such uses can include but are not limited to: (1) use as
a calibration standard for quantitating the activities of candidate
EPO-R agonists in a variety of functional assays; (2) use as
blocking reagents in random peptide screening, i.e., in looking for
new families of EPO-R peptide ligands, the peptides can be used to
block recovery of EPO peptides of the present invention; (3) use in
co-crystallization with EPO-R, i.e., crystals of the peptides of
the present invention bound to the EPO-R may be formed, enabling
determination of receptor/peptide structure by X-ray
crystallography; (4) use to measure the capacity of erythrocyte
precursor cells induce globin synthesis and heme complex synthesis,
and to increase the number of ferritin receptors, by initiating
differentiation; (5) use to maintain the proliferation and growth
of EPO-dependent cell lines, such as the FDCP-1-mEPO-R and the TF-1
cell lines; (6) use related to labeling the peptides of the
invention with a radioactive chromophore; and (7) other research
and diagnostic applications wherein the EPO-R is preferably
activated or such activation is conveniently calibrated against a
known quantity of an EPO-R agonist, and the like.
[0131] In yet another aspect of the present invention, methods of
treatment and manufacture of a medicament are provided. The peptide
compounds of the invention may be administered to warm blooded
animals, including humans, to simulate the binding of EPO to the
EPO-R in vivo. Thus, the present invention encompasses methods for
therapeutic treatment of disorders associated with a deficiency of
EPO, which methods comprise administering a peptide of the
invention in amounts sufficient to stimulate the EPO-R and thus,
alleviate the symptoms associated with a deficiency of EPO in vivo.
For example, the peptides of this invention will find use in the
treatment of renal insufficiency and/or end-stage renal
failure/dialysis; chronic kidney disease, anemia associated with
AIDS; anemia associated with chronic inflammatory diseases (for
example, rheumatoid arthritis and chronic bowel inflammation) and
autoimmune disease; and for boosting the red blood count of a
patient prior to surgery. Other disease states, disorders, and
states of hematologic irregularity that may be treated by
administration of the peptides of this invention include:
beta-thalassemia; cystic fibrosis; pregnancy and menstrual
disorders; early anemia of prematurity; spinal cord injury; space
flight; acute blood loss; aging; stroke, ischemia (both CNS and
cardiac); and various neoplastic disease states accompanied by
abnormal erythropoiesis.
[0132] In other embodiments, the peptide compounds of the invention
may be used for the treatment of disorders which are not
characterized by low or deficient red blood cells, for example as a
pretreatment prior to transfusions. In addition, administration of
the compounds of this invention can result in a decrease in
bleeding time and thus, will find use in the administration to
patients prior to surgery or for indications wherein bleeding is
expected to occur. In addition, the compounds of this invention
will find use in the activation of megakaryocytes.
[0133] Since EPO has been shown to have a mitogenic and chemotactic
effect on vascular endothelial cells as well as an effect on
central cholinergic neurons [see, e.g., Amagnostou, et al. (1990)
Proc. Natl. Acad. Sci. USA 87:5978-5982 and Konishi, et al. (1993)
Brain Res. 609:29-35], the compounds of this invention will also
find use for the treatment of a variety of vascular disorders, such
as: promoting wound healing; promoting growth of collateral
coronary blood vessels (such as those that may occur after
myocardial infarction); trauma treatment; and post-vascular graft
treatment. The compounds of this invention will also find use for
the treatment of a variety of neurological disorders, generally
characterized by low absolute levels of acetylcholine or low
relative levels of acetylcholine as compared to other neuroactive
substances e.g., neurotransmitters.
Pharmaceutical Compositions
[0134] In yet another aspect of the present invention,
pharmaceutical compositions of the above EPO-R agonist peptide
compounds are provided. Conditions alleviated or modulated by the
administration of such compositions include those indicated above.
Such pharmaceutical compositions may be for administration by oral,
parenteral (intramuscular, intraperitoneal, intravenous (IV) or
subcutaneous injection), transdermal (either passively or using
iontophoresis or electroporation), transmucosal (nasal, vaginal,
rectal, or sublingual) routes of administration or using
bioerodible inserts and can be formulated in dosage forms
appropriate for each route of administration. In general,
comprehended by the invention are pharmaceutical compositions
comprising effective amounts of an EPO-R agonist peptide, or
derivative products, of the invention together with
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/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 20, Tween 80, Polysorbate 80),
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 liposomes.
Hylauronic acid may also be used. 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., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein
incorporated by reference. The compositions may be prepared in
liquid form, or may be in dried powder (e.g., lyophilized)
form.
Oral Delivery
[0135] Contemplated for use herein are oral solid dosage forms,
which are described generally in Remington's Pharmaceutical
Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at
Chapter 89, which is herein incorporated by reference. Solid dosage
forms include tablets, capsules, pills, troches or lozenges,
cachets, pellets, powders, or granules. Also, liposomal or
proteinoid encapsulation may be used to formulate the present
compositions (as, for example, proteinoid microspheres reported in
U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and
the liposomes may be derivatized with various polymers (e.g., U.S.
Pat. No. 5,013,556). A description of possible solid dosage forms
for the therapeutic is given by Marshall, K. In: Modern
Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10,
1979, herein incorporated by reference. In general, the formulation
will include the EPO-R agonist peptides (or chemically modified
forms thereof) and inert ingredients which allow for protection
against the stomach environment, and release of the biologically
active material in the intestine.
[0136] Also contemplated for use herein are liquid dosage forms for
oral administration, including pharmaceutically acceptable
emulsions, solutions, suspensions, and syrups, which may contain
other components including inert diluents; adjuvants such as
wetting agents, emulsifying and suspending agents; and sweetening,
flavoring, and perfuming agents.
[0137] The peptides may be chemically modified so that oral
delivery of the derivative is efficacious. Generally, the chemical
modification contemplated is the attachment of at least one moiety
to the component 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 component or components and increase in
circulation time in the body. As discussed above, PEGylation is a
preferred chemical modification for pharmaceutical usage. Other
moieties that may be used include: propylene glycol, copolymers of
ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline,
poly-1,3-dioxolane and poly-1,3,6-trioxocane [see, e.g., Abuchowski
and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as
Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York,
N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.
4:185-189].
[0138] For oral formulations, the location of release may be the
stomach, the small intestine (the duodenum, the jejunem, or the
ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
peptide (or derivative) or by release of the peptide (or
derivative) beyond the stomach environment, such as in the
intestine.
[0139] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0140] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic (i.e. powder), for liquid
forms a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0141] The peptide (or derivative) 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 administration could also be as a powder,
lightly compressed plugs, or even as tablets. These therapeutics
could be prepared by compression.
[0142] Colorants and/or flavoring agents may also be included. For
example, the peptide (or derivative) may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0143] One may dilute or increase the volume of the peptide (or
derivative) with an inert material. These diluents could include
carbohydrates, especially mannitol, .alpha.-lactose, anhydrous
lactose, cellulose, sucrose, modified dextrans and starch. Certain
inorganic salts may be 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.
[0144] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates 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. The disintegrants may also be insoluble cationic exchange
resins. Powdered gums may be used as disintegrants and as binders
and can include powdered gums such as agar, Karaya or tragacanth.
Alginic acid and its sodium salt are also useful as
disintegrants.
[0145] Binders may be used to hold the peptide (or derivative)
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 cellulose (EC) and
carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the peptide (or derivative).
[0146] An antifrictional agent may be included in the formulation
of the peptide (or derivative) to prevent sticking during the
formulation process. Lubricants may be used as a layer between the
peptide (or derivative) and the die wall, and these can include but
are not limited to; stearic acid including its magnesium and
calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin,
vegetable oils and waxes. Soluble lubricants may also be used such
as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene
glycol of various molecular weights, Carbowax 4000 and 6000.
[0147] 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.
[0148] To aid dissolution of the peptide (or derivative) 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 20, 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.
[0149] Additives which potentially enhance uptake of the peptide
(or derivative) are for instance the fatty acids oleic acid,
linoleic acid and linolenic acid.
[0150] Controlled release oral formulations may be desirable. The
peptide (or derivative) 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. Some enteric coatings also have a delayed
release effect. Another form of a controlled release 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.
[0151] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The peptide (or derivative) 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 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.
[0152] 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.
Parenteral Delivery
[0153] Preparations according to this invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized by, for example, filtration through
a bacteria retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured using
sterile water, or some other sterile injectable medium, immediately
before use.
Rectal or Vaginal Delivery
[0154] Compositions for rectal or vaginal administration are
preferably suppositories which may contain, in addition to the
active substance, excipients such as cocoa butter or a suppository
wax. Compositions for nasal or sublingual administration are also
prepared with standard excipients well known in the art.
Pulmonary Delivery
[0155] Also contemplated herein is pulmonary delivery of the EPO-R
agonist peptides (or derivatives thereof). The peptide (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the blood stream
[see, e.g., Adjei, et al. (1990) Pharmaceutical Research 7:565-569;
Adjei, et al. (1990) Int. J. Pharmaceutics 63:135-144 (leuprolide
acetate); Braquet, et al. (1989) J. Cardiovascular Pharmacology
13(sup5):143-146 (endothelin-1); Hubbard, et al. (1989) Annals of
Internal Medicine, Vol. III, pp. 206-212 (.alpha.1-antitrypsin);
Smith, et al. (1989) J. Clin. Invest. 84:1145-1146
(.alpha.-1-proteinase); Oswein, et al. (1990) "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery II
Keystone, Colorado (recombinant human growth hormone); Debs, et al.
(1988) J. Immunol. 140:3482-3488 (interferon-.gamma. and tumor
necrosis factor .alpha.); and U.S. Pat. No. 5,284,656 to Platz, et
al. (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569 to Wong, et al.
[0156] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
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 (Mallinckrodt Inc., St.
Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products,
Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc.,
Research Triangle Park, N.C.); and the Spinhaler powder inhaler
(Fisons Corp., Bedford, Mass.).
[0157] All such devices require the use of formulations suitable
for the dispensing of peptide (or derivative). Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants and/or carriers useful in therapy.
Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is contemplated.
Chemically modified peptides may also be prepared in different
formulations depending on the type of chemical modification or the
type of device employed.
[0158] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise peptide (or derivative)
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 reduce or prevent
surface induced aggregation of the peptide (or derivative) caused
by atomization of the solution in forming the aerosol.
[0159] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the peptide
(or derivative) 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.
[0160] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing peptide (or
derivative) and may also include a bulking agent, such as lactose,
sorbitol, sucrose, or mannitol in amounts which facilitate
dispersal of the powder from the device, e.g., 50 to 90% by weight
of the formulation. The peptide (or derivative) should most
advantageously be prepared in particulate form with an average
particle size of less than 10 mm (or microns), most preferably 0.5
to 5 mm, for most effective delivery to the distal lung.
Nasal Delivery
[0161] Nasal delivery of the EPO-R agonist peptides (or
derivatives) is also contemplated. Nasal delivery allows the
passage of the peptide to the 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.
[0162] Other penetration-enhancers used to facilitate nasal
delivery are also contemplated for use with the peptides of the
present invention (such as described in International Patent
Publication No. WO 2004056314, filed Dec. 17, 2003, incorporated
herein by reference in its entirety).
Dosages
[0163] For all of the peptide compounds, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age, and general health of the recipient, will be able to ascertain
proper dosing. The selected dosage depends upon the desired
therapeutic effect, on the route of administration, and on the
duration of the treatment desired. Generally dosage levels of 0.001
to 10 mg/kg of body weight daily are administered to mammals.
Generally, for intravenous injection or infusion, dosage may be
lower.
[0164] In certain embodiments, any one of the peptides of the
present invention may be used to treat individuals with renal
failure prior to dialysis or during dialysis (pre-dialysis or
dialysis patients). The therapeutic dose range in this embodiment
can be 0.025 to 0.2 milligrams (mg) of compound per 1 kilogram (kg)
of body weight of the individual (0.025-0.2 mg/kg). More
particularly, the dose range of 0.05-0.1 mg/kg would be preferred.
Furthermore, a physician may initially use escalating dosages,
starting at 0.025 mg/kg, and then titrate the dosage at
approximately 0.025 mg/kg increments for each individual being
treated based on their individual hemoglobin responses. Thus, the
physician may titrate the dosage for each individual until an
adequate hemoglobin response is achieved. In the case of
individuals who are pre-dialysis or dialysis patients, the adequate
hemoglobin response would be to approximately attain normal
hemoglobin levels (14-15 g/dL) or another hemoglobin level as
determined by the physician. In this embodiment, the
pharmacologically active dose (PAD) for each individual
pre-dialysis or dialysis patient is expected to be 0.067-0.075
mg/kg. An advantage of this embodiment is expected to be a lower
dosing frequency of once every three to four weeks for each
individual patient instead of weekly as is the case for other
current erythropoiesis stimulating agents (ESAs). Many routes of
administration may be used (oral, IV, etc. as described above). A
preferred route of administration for dialysis patients would be
intravenously. A preferred route of administration for pre-dialysis
patients would be subcutaneously. In other certain embodiments, one
of the compounds described above may be used to treat individuals
with anemia associated with malignancies (oncology patients). The
therapeutic dose range in this embodiment is expected to be three
to five times the range for pre-dialysis or dialysis patients
(i.e., 0.075-0.5 mg/kg or, alternatively, between 0.05-0.5 mg/kg).
More particularly, the dose range of 0.2-0.4 mg/kg would be
preferred. As above, the physician treating the oncology patients
may titrate the dosage, starting at 0.075 mg/kg or, alternatively,
at 0.05 mg/kg, and increasing at 0.075 mg/kg or, alternatively, at
0.05 mg/kg increments until an adequate hemoglobin response is
attained. The PAD for each individual oncology patient is expected
to be approximately 0.25 mg/kg. Again, the advantage of less
frequent dosage of every three to four weeks is expected for each
individual patient. Furthermore, other advantages for oncology
patients is the dosage may be administered prior to chemotherapy
(for example, 3-5 days beforehand) or co-administered with
chemotherapy to prevent the decline in hemoglobin during the lag
phase between reticulocyte stimulation and hemoglobin rise. Many
routes of administration may be used (oral, IV, etc. as described
above). Subcutaneous administration would be a preferred route of
administration for oncology patients. Preferred compounds for use
in treating pre-dialysis, dialysis, or oncology patients include
those shown below.
[0165] Carbamate linkage, no sarcosine, and with the range of PEG
weights (here showing SEQ ID NO: 1):
##STR00046##
[0166] Carbamate linkage, no sarcosine, and preferred PEG weights
(here showing SEQ ID NO: 1):
##STR00047## ##STR00048## ##STR00049##
[0167] Carbamate linkage, with sarcosine, and the preferred PEG
weights (here showing SEQ ID NO: 2):
##STR00050## ##STR00051## ##STR00052##
[0168] Amide linkage, no sarcosine, and with the range of PEG
weights (here showing SEQ ID NO: 1):
##STR00053##
[0169] Amide linkage, no sarcosine, and the preferred PEG weights
(here showing SEQ ID NO: 1):
##STR00054## ##STR00055## ##STR00056##
[0170] Amide linkage, with sarcosine, and range of PEG weights
(here showing SEQ ID NO: 2):
##STR00057##
[0171] Amide linkage, sarcosine, and the preferred PEG weights
(here showing SEQ ID NO: 2):
##STR00058## ##STR00059## ##STR00060##
[0172] The peptides of the present invention (or their derivatives)
may be administered in conjunction with one or more additional
active ingredients or pharmaceutical compositions.
EXAMPLES
[0173] The present invention is next described by means of the
following examples. However, the use of these and other examples
anywhere in the specification is illustrative only, and in no way
limits the scope and meaning of the invention or of any exemplified
form. Likewise, the invention is not limited to any particular
preferred embodiments described herein. Indeed, many modifications
and variations of the invention may be apparent to those skilled in
the art upon reading this specification, and can be made without
departing from its spirit and scope. The invention is therefore to
be limited only by the terms of the appended claims, along with the
full scope of equivalents to which the claims are entitled.
Example 1
Synthesis of EPO-R Agonist Peptide Dimers by Solid Phase
Synthesis
[0174] Step 1--Synthesis of Cbz-TAP:
[0175] A solution containing the commercially available diamine
("TAP" from Aldrich Chemical Co.) (10 g, 67.47 mmol) in anhydrous
DCM (100 ml) was cooled to 0.degree. C. A solution of benzyl
chloroformate (4.82 ml, 33.7 mmol) in anhydrous DCM (50 ml) was
added slowly through a dropping funnel over a period of 6-7 h,
maintaining the temperature of the reaction mixture at 0.degree. C.
throughout, then allowed to warm to room temperature
(.about.25.degree. C.). After a further 16 h, the DCM was removed
under vacuum and the residue partitioned between 3N HCl and ether.
The aqueous layers were collected and neutralized with 50% aq. NaOH
to pH 8-9 and extracted with ethyl acetate. The ethyl acetate layer
was dried to over anhydrous Na.sub.2SO.sub.4, and then concentrated
under vacuum to provide the crude mono-Cbz-TAP (5 g, about 50%
yield). This compound was used for the next reaction without any
further purification.
##STR00061##
[0176] Step 2--Synthesis of Cbz-TAP-Boc:
[0177] To a vigorously stirred suspension of the Cbz-TAP (5 g, 17.7
mmol) in hexane (25 ml) was added Boc.sub.2O (3.86 g, 17.7 mmol)
and stirring continued at RT overnight. The reaction mixture was
diluted with DCM (25 ml) and washed with 10% aq. citric acid
(2.times.), water (2.times.) and brine. The organic layer was dried
over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The
crude product (yield 5 g) was used directly in the next
reaction.
##STR00062##
[0178] Step 3--Synthesis of Boc-TAP:
[0179] The crude product from the previous reaction was dissolved
in methanol (25 ml) and hydrogenated in presence of 5% Pd on Carbon
(5% w/W) under balloon pressure for 16 hrs. The mixture was
filtered, washed with methanol and the filtrate concentrated in
vacuo to provide the crude H-TAP-Boc product (yield 3.7 g). The
overall approximate yield of Boc-TAP after Steps 1-3 was 44%
(calculated based on the amount of Cbz-Cl used.)
##STR00063##
[0180] Step 4--Synthesis of TentaGel-Linker:
[0181] TentaGel bromide (2.5 g, 0.48 mmol/g, from Rapp Polymere,
Germany), phenolic linker (5 equivalent, and K.sub.2CO.sub.3 (5
equivalent) were heated in 20 mL of DMF to 70.degree. C. for 14 h.
After cooling to room temperature, the resin was washed (0.1 N HCl,
water, ACN, DMF, MeOH) and dried to give an amber-colored
resin.
##STR00064##
[0182] Step 5--Synthesis of TentaGel-linker-TAP(Boc):
[0183] 2.5 gm of the resin from above and H-TAP-Boc (1.5 gm, 5 eq.)
and glacial AcOH (34 .mu.l, 5 eq.) was taken in a mixture of 1:1
MeOH-THF and shaken overnight. A 1M solution of sodium
cyanoborohydride (5 eq) in THF was added to this and shaken for
another 7 hrs. The resin was filtered washed (DMF, THF, 0.1 N HCl,
water, MeOH) and dried. A small amount of the resin was benzoylated
with Bz--Cl and DIEA in DCM and cleaved with 70% TFA-DCM and
checked by LCMS and HPLC.
##STR00065##
[0184] Step 6--Synthesis of TentaGel-Linker-TAP-Lys:
[0185] The resin from above was treated with a activated solution
of Fmoc-Lys(Fmoc)-OH (prepared from 5 eq. of amino acid and 5 eq.
of HATU dissolved at 0.5 M in DMF, followed by the addition of 10
eq. of DIEA) and allowed to gently shake 14 h. The resin was washed
(DMF, THF, DCM, MeOH) and dried to yield the protected resin.
Residual amine groups were capped by treating the resin with a
solution of 10% acetic anhydride, 20% pyridine in DCM for 20
minutes, followed by washing as above. The Fmoc groups are removed
by gently shaking the resin in 30% piperidine in DMF for 20
minutes, followed by washing (DMF, THF, DCM, MeOH) and drying.
##STR00066##
[0186] Step 7--Synthesis of
TentaGel-Linker-TAP-Lys(Peptide).sub.2:
[0187] The resin from above was subjected to repeated cycles of
Fmoc-amino acid couplings with HBTU/HOBt activation and Fmoc
removal with piperidine to build both peptide chains
simultaneously. This was conveniently carried out on an ABI 433
automated peptide synthesizer available from Applied Biosystems,
Inc. After the final Fmoc removal, the terminal amine groups were
acylated with acetic anhydride (10 eq.) and DIEA (20 eq.) in DMF
for 20 minutes, followed by washing as above.
##STR00067##
[0188] Step 8--Cleavage from Resin:
[0189] The resin from above was suspended in a solution of TFA
(82.5%), phenol (5%), ethanedithiol (2.5%), water (5%), and
thioanisole (5%) for 3 h at room temperature. Alternative cleavage
cocktails such as TFA (95%), water (2.5%), and triisopropylsilane
(2.5%) can also be used. The TFA solution was cooled to 5.degree.
C. and poured into Et.sub.2O to precipitate the peptide. Filtration
and drying under reduced pressure gave the desired peptide.
Purification via preparative HPLC with a C18 column afforded the
pure peptide.
##STR00068##
[0190] Step 9--Oxidation of Peptides to Form Intramolecular
Disulfide Bonds:
[0191] The peptide dimer was dissolved in 20% DMSO/water (1 mg dry
weight peptide/mL) and allowed to stand at room temperature for 36
h. The peptide was purified by loading the reaction mixture onto a
C18 HPLC column (Waters Delta-Pak C18, 15 micron particle size, 300
angstrom pore size, 40 mm.times.200 mm length), followed by a
linear ACN/water/0.01% TFA gradient from 5 to 95% ACN over 40
minutes, Lypholization of the fractions containing the desired
peptide affords the product as a fluffy white solid.
##STR00069##
[0192] Step 10--PEGylation of the terminal --NH.sub.2 Group:
[0193] PEGylation Via a Carbamate Bond:
[0194] The peptide dimer was mixed with 1.5 eq. (mole basis) of
activated PEG species (mPEG-NPC from NOF Corp. Japan) in dry DMF to
afford a clear solution. After 5 minutes 4 eq of DIEA was added to
above solution. The mixture was stirred at ambient temperature 14
h, followed by purification with C18 reverse phase HPLC. The
structure of PEGylated peptide was confirmed by MALDI mass. The
purified peptide was also subjected to purification via cation ion
exchange chromatography as outlined below. The below scheme shows
mPEG-NPC PEGylation using SEQ ID NO: 3.
##STR00070##
[0195] PEGylation Via an Amide Bond:
[0196] The peptide dimer is mixed with 1.5 eq. (mole basis) of 1
eq. activated PEG species (PEG-SPA-NHS from Shearwater Corp, USA)
in dry DMF to afford a clear solution. After minutes 10 eq of DIEA
is added to above solution. The mixture is stirred at ambient
temperature 2 h, followed by purification with C18 reverse phase
HPLC. The structure of PEGylated peptide was confirmed by MALDI
mass. The purified peptide was also subjected to purification via
cation ion exchange chromatography as outlined below. The below
scheme shows PEG-SPA-NHS PEGylation using SEQ ID NO: 3.
##STR00071##
[0197] Step 11--Ion Exchange Purification:
[0198] Several exchange supports were surveyed for their ability to
separate the above peptide-PEG conjugate from unreacted (or
hydrolyzed) PEG, in addition to their ability to retain the
starting dimeric peptides. The ion exchange resin (2-3 g) was
loaded into a 1 cm column, followed by conversion to the sodium
form (0.2 N NaOH loaded onto column until elutant was pH 14, ca. 5
column volumes), and than to the hydrogen form (eluted with either
0.1 N HCl or 0.1 M HOAc until elutant matched load pH, ca. 5 column
volumes), followed by washing with 25% ACN/water until pH 6. Either
the peptide prior to conjugation or the peptide-PEG conjugate was
dissolved in 25% ACN/water (10 mg/mL) and the pH adjusted to <3
with TFA, then loaded on the column. After washing with 2-3 column
volumes of 25% ACN/water and collecting 5 mL fractions, the peptide
was released from the column by elution with 0.1 M NH.sub.4OAc in
25% ACN/water, again collecting 5 mL fractions. Analysis via HPLC
revealed which fractions contained the desired peptide, Analysis
with an Evaporative Light-Scattering Detector (ELSD) indicated that
when the peptide was retained on the column and was eluted with the
NH.sub.4OAc solution (generally between fractions 4 and 10), no
non-conjugated PEG was observed as a contaminant. When the peptide
eluted in the initial wash buffer (generally the first 2
fractions), no separation of desired PEG-conjugate and excess PEG
was observed.
[0199] The following columns successfully retained both the peptide
and the peptide-PEG conjugate, and successfully purified the
peptide-PEG conjugate from the unconjugated peptide:
TABLE-US-00001 TABLE 1 Ion Exchange Resins Support Source Mono S HR
5/5 strong cation Amersham Biosciences exchange pre-loaded column
SE53 Cellulose, microgranular Whatman strong cation exchange
support SP Sepharose Fast Flow strong Amersham Biosciences cation
exchange support
Example 2
Synthesis of EPO-R Agonist Peptide Dimers by Fragment
Condensation
[0200] Step 1--Synthesis of (Cbz).sub.2-Lys:
[0201] Lysine is reacted under standard conditions with a solution
of benzyl chloroformate to obtain lysine protected at its two amino
groups with a Cbz group.
##STR00072##
[0202] Step 2--Synthesis of Boc-TAP:
[0203] Boc-TAP is synthesized as described in Steps 1 through 3 of
Example 1.
[0204] Step 3--Coupling of (Cbz).sub.2-Lys and Boc-TAP:
[0205] (Cbz).sub.2-Lys and Boc-TAP are coupled under standard
coupling conditions to obtain (Cbz).sub.2-Lys-TAP-Boc.
##STR00073##
[0206] Step 4-Lys-TAP-Boc:
[0207] The crude product from the previous reaction is dissolved in
methanol (25 ml) and hydrogenated in presence of 5% Pd on Carbon
(5% w/W) under balloon pressure for 16 hrs. The mixture is
filtered, washed with methanol and the filtrate concentrated in
vacuo to provide the crude Lys-TAP-Boc product
##STR00074##
[0208] Step 5--Synthesis of Peptide Monomers by Fragment
Condensation:
[0209] Four peptide fragments of the peptide monomer sequence are
synthesized by standard techniques. These partially protected
fragments are then subjected to two independent rounds of coupling.
In the first round, the N-terminal half of the monomer is formed by
coupling two of the peptide fragments, while the C-terminal half of
the monomer is formed by coupling the other two of the peptide
fragments. In the second round of coupling, the N-terminal and
C-terminal halves are coupled to form the fully protected monomer.
The monomer is then OBn-deprotected by standard techniques.
##STR00075##
[0210] Step 6--Oxidation of Peptide Monomers to Form Intramolecular
Disulfide Bonds:
[0211] The OBn-deprotected condensed peptide monomers (SEQ ID NO:
12) are then oxidized with under iodide to form intramolecular
disulfide bonds between the Acm-protected cysteine residues of the
monomers.
##STR00076##
[0212] Step 7--Coupling of Lys-TAP-Boc to Oxidized OBn-Deprotected
Monomers to Form a Peptide Dimer:
[0213] Lys-TAP-Boc is coupled to a two-fold molar excess of the
oxidized OBn-deprotected monomers under standard conditions to form
a peptide dimer. The peptide dimer is then deprotected under
standard conditions.
##STR00077##
[0214] Step 8--PEGylation of Deprotected Dimer:
[0215] The deprotected peptide dimer is then PEGylated as described
in Step 10 of Example 1.
[0216] Step 9--Ion Exchange Purification:
[0217] The PEGylated peptide dimer is then purified as described in
Step 11 of Example 1.
Example 3
In Vitro Activity Assays
[0218] This example describes various in vitro assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. The results for these assays demonstrate
that the novel peptides of this invention bind to EPO-R and
activate EPO-R signaling. Moreover, the results for these assays
show that the novel peptide compositions exhibit a surprising
increase in EPO-R binding affinity and biological activity compared
to EPO mimetic peptides that have been previously described.
[0219] EPO-R agonist peptide dimers are prepared according to the
methods provided in Example 1 or Example 2. The potency of these
peptide dimers is evaluated using a series of in vitro activity
assays, including: a reporter assay, a proliferation assay, a
competitive binding assay, and a C/BFU-e assay. These four assays
are described in further detail below.
[0220] The results of these in vitro activity assays are summarized
in Table 2.
I. Reporter Assay
[0221] This assay is based upon a on a murine pre-B-cell line
derived reporter cell, Baf3/EpoR/GCSFR fos/lux. This reporter cell
line expresses a chimeric receptor comprising the extra-cellular
portion of the human EPO receptor to the intra-cellular portion of
the human GCSF receptor. This cell line is further transfected with
a fos promoter-driven luciferase reporter gene construct.
Activation of this chimeric receptor through addition of
erythropoietic agent results in the expression of the luciferase
reporter gene, and therefore the production of light upon addition
of the luciferase substrate luciferin. Thus, the level of EPO-R
activation in such cells may be quantitated via measurement of
luciferase activity.
[0222] The Baf3/EpoR/GCSFR fos/lux cells are cultured in DMEM/F12
medium (Gibco) supplemented with 10% fetal bovine serum (FBS;
Hyclone), 10% WEHI-3 supernatant (the supernatant from a culture of
WEHI-3 cells, ATCC # TIB-68), and penicillin/streptomycin.
Approximately 18 h before the assay, cells are starved by
transferring them to DMEM/F12 medium supplemented with 10% FBS and
0.1% WEHI-3 supernatant. On the day of assay, cells are washed once
with DMEM/F12 medium supplemented with 10% FBS (no WEHI-3
supernatant), then 1.times.10.sup.6 cells/mL are cultured in the
presence of a known concentration of test peptide, or with EPO(R
& D Systems Inc., Minneapolis, Minn.) as a positive control, in
DMEM/F12 medium supplemented with 10% FBS (no WEHI-3 supernatant).
Serial dilutions of the test peptide are concurrently tested in
this assay. Assay plates are incubated for 4 h at 37.degree. C. in
a 5% CO.sub.2 atmosphere, after which luciferin (Steady-Glo;
Promega, Madison, Wis.) is added to each well. Following a 5-minute
incubation, light emission is measured on a Packard Topcount
Luminometer (Packard Instrument Co., Downers Grove, Ill.). Light
counts are plotted relative to test peptide concentration and
analysed using Graph Pad software. The concentration of test
peptide that results in a half-maximal emission of light is
recorded as the EC50 [See Table 2: Reporter EC50].
2. Proliferation Assay
[0223] This assay is based upon a murine pre-B-cell line, Baf3,
transfected to express human EPO-R. Proliferation of the resulting
cell line, BaF3/Gal4/Elk/EPOR, is dependent on EPO-R activation.
The degree of cell proliferation is quantitated using MTT, where
the signal in the MTT assay is proportional to the number of viable
cells.
[0224] The BaF3/Gal4/Elk/EPOR cells are cultured in spinner flasks
in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and
2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells are starved
overnight, in a spinner flask at a cell density of 1.times.10.sup.6
cells/ml, in DMEM/F12 medium supplemented with 10% FBS and 0.1%
WEHI-3 supernatant. The starved cells are then washed twice with
Dulbecco's PBS (Gibco), and resuspended to a density of
1.times.10.sup.6 cells/ml in DMEM/F12 supplemented with 10% FBS (no
WEHI-3 supernatant). 50 .mu.L aliquots (.about.50,000 cells) of the
cell suspension are then plated, in triplicate, in 96 well assay
plates. 50 .mu.L aliquots of dilution series of test EPO mimetic
peptides, or 50 .mu.L EPO(R & D Systems Inc., Minneapolis,
Minn.) or Aranesp.TM. (darbepoeitin alpha, an ERO-R agonist
commercially available from Amgen) in DMEM/F12 media supplemented
with 10% FBS (no WEHI-3 supernatant I) are added to the 96 well
assay plates (final well volume of 1004). For example, 12 different
dilutions may be tested where the final concentration of test
peptide (or control EPO peptide) ranges from 810 pM to 0.0045 pM.
The plated cells are then incubated for 48 h at 37.degree. C. Next,
10 .mu.L of MTT (Roche Diagnostics) is added to each culture dish
well, and then allowed to incubate for 4 h. The reaction is then
stopped by adding 10% SDS+0.01N HCl. The plates are then incubated
overnight at 37.degree. C. Absorbance of each well at a wavelength
of 595 nm is then measured by spectrophotometry. Plots of the
absorbance readings versus test peptide concentration are
constructed and the EC50 calculated using Graph Pad software. The
concentration of test peptide that results in a half-maximal
absorbance is recorded as the EC50 [See Table 2: Proliferation
EC50].
3. Competitive Binding Assay
[0225] Competitive binding calculations are made using an assay in
which a light signal is generated as a function of the proximity of
two beads: a streptavidin donor bead bearing a biotinylated
EPO-R-binding peptide tracer and an acceptor bead to which is bound
EPO-R. Light is generated by non-radiative energy transfer, during
which a singlet oxygen is released from a first bead upon
illumination, and contact with the released singlet oxygen causes
the second bead to emit light. These bead sets are commercially
available (Packard). Bead proximity is generated by the binding of
the EPO-R-binding peptide tracer to the EPO-R. A test peptide that
competes with the EPO-R-binding peptide tracer for binding to EPO-R
will prevent this binding, causing a decrease in light
emission.
[0226] In more detail the method is as follows: Add 4 .mu.L of
serial dilutions of the test EPO-R agonist peptide, or positive or
negative controls, to wells of a 384 well plate. Thereafter, add 2
.mu.L/well of receptor/bead cocktail. Receptor bead cocktail
consists of: 15 .mu.L of 5 mg/ml streptavidin donor beads
(Packard), 15 .mu.L of 5 mg/ml monoclonal antibody ab179 (this
antibody recognizes the portion of the human placental alkaline
phosphatase protein contained in the recombinant EPO-R), protein
A-coated acceptor beads (protein A will bind to the ab179 antibody;
Packard), 112.5 .mu.L of a 1:6.6 dilution of recombinant EPO-R
(produced in Chinese Hamster Ovary cells as a fusion protein to a
portion of the human placental alkaline phosphatase protein which
contains the ab179 target epitope) and 607.54 of Alphaquest buffer
(40 mM HEPES, pH 7.4; 1 mM MgCl.sub.2; 0.1% BSA, 0.05% Tween 20).
Tap to mix. Add 2 .mu.L/well of the biotinylated EPO-R-binding
peptide tracer (30 nM final concentration). The peptide tracer, an
EPO-R binding peptide (see in the tables "Reporter EC50 (pM)"), is
made according to the methods described in Example 1, with sequence
Biotin-GGLYACHMGPITWVCQPLRG (SEQ ID NO: 4).
##STR00078##
[0227] Centrifuge 1 min to mix. Seal plate with Packard Top Seal
and wrap in foil. Incubate overnight at room temperature. After 18
hours read light emission using an AlphaQuest reader (Packard).
Plot light emission vs. concentration of peptide and analyze with
Graph Pad or Excel.
[0228] The concentration of test peptide that results in a 50%
decrease in light emission, relative to that observed without test
peptide, is recorded as the IC50 [See Table 2: AQ IC50].
4. C/BFU-e Assay
[0229] EPO-R signaling stimulates the differentiation of bone
marrow stem cells into proliferating red blood cell precursors.
This assay measures the ability of test peptides to stimulate the
proliferation and differentiation of red blood cell precursors from
primary human bone marrow pluripotent stem cells.
[0230] For this assay, serial dilutions of test peptide are made in
IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These
serial dilutions, or positive control EPO peptide, are then added
to methylcellulose to give a final volume of 1.5 mL. The
methylcellulose and peptide mixture is then vortexed thoroughly.
Aliquots (100,000 cells/mL) of human, bone marrow derived CD34+
cells (Poietics/Cambrex) are thawed. The thawed cells are gently
added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50 mL tube.
Next, 40-50 mL IMDM medium is added gently to cells: the medium is
added drop by drop along the side of the 50 mL tube for the first
10 mL, and then the remaining volume of medium is slowly dispensed
along the side of the tube. The cells are then spun at 900 rpm for
20 min, and the media removed carefully by gentle aspiration. The
cells are resuspended in 1 ml of IMDM medium and the cell density
per mL is counted on hemacytometer slide (10 .mu.L aliquot of cell
suspension on slide, and cell density is the average count X 10,000
cells/ml). The cells are then diluted in IMDM medium to a cell
density of 15,000 cells/mL. A 100 .mu.L of diluted cells is then
added to each 1.5 mL methyl cellulose plus peptide sample (final
cell concentration in assay media is 1000 cells/mL), and the
mixture is vortexed. Allow the bubbles in the mixture to disappear,
and then aspirate 1 mL using blunt-end needle. Add 0.25 mL
aspirated mixture from each sample into each of 4 wells of a
24-well plate (Falcon brand). Incubate the plated mixtures at
37.degree. C. under 5% CO.sub.2 in a humid incubator for 14 days.
Score for the presence of erythroid colonies using a phase
microscope (5.times.-10.times. objective, final magnification of
100.times.). The concentration of test peptide at which the number
of formed colonies is 90% of maximum, relative to that observed
with the EPO positive control, is recorded as the EC90 [See Table
2: C/BFU-e EC90].
5. Radioligand Competitive Binding Assay
[0231] An alternative radioligand competition binding assay can
also be used to measure IC.sub.50 values of peptides in this
invention. This assay measures binding of .sup.125I-EPO to EPOr.
The assay is preferably performed according to the following
exemplary protocol:
[0232] A. Materials
TABLE-US-00002 Recombinant Identification: Recombinant Human EPO
R/Fc Chimera Human Supplier: R&D Systems (Minneapolis, MN, US)
EPO R/Fc Catalog number: 963-ER Chimera Lot number: EOK033071
Storage: 4.degree. C. Iodinated Identification:
(3[.sup.125I]iodotyrosyl)Erythropoietin, human recombinant
recombinant, high specific activity, 370 kBq, 10 .mu.Ci human
Supplier: Amersham Biosciences (Piscataway, NJ, US) Erythropoietin
Catalog number: IM219-10 .mu.Ci Lot number: Storage: 4.degree. C.
Protein-G Identification: Protein-G Sepharose 4 Fast Flow Sepharose
Supplier: Amersham Biosciences (Piscataway, NJ, US) Catalog number
17-0618-01 Lot number: Storage: 4.degree. C. Assay Phosphate
Buffered Saline (PBS), pH 7.4, containing 0.1% Buffer Bovine Serum
Albumin and 0.1% Sodium Azide Storage: 4.degree. C.
[0233] B. Determination of Appropriate Receptor Concentration.
[0234] One 50 .mu.g vial of lyophilized recombinant EPOr
extracellular domain fused to the Fc portion of human IgG1 is
reconstituted in 1 mL of assay buffer. To determine the correct
amount of receptor to use in the assay, 100 .mu.L serial dilutions
of this receptor preparation are combined with approximately 20,000
cpm in 200 .mu.L of iodinated recombinant human Erythropoietin
(.sup.125I-EPO) in 12.times.75 mm polypropylene test tubes. Tubes
are capped and mixed gently at 4.degree. C. overnight on a LabQuake
rotating shaker.
[0235] The next day, 50 .mu.L of a 50% slurry of Protein-G
Sepharose is added to each tube. Tubes are then incubated for 2
hours at 4.degree. C., mixing gently. The tubes are then
centrifuged for 15 min at 4000 RPM (3297.times.G) to pellet the
protein-G sepharose. The supernatants are carefully removed and
discarded. After washing 3 times with 1 mL of 4.degree. C. assay
buffer, the pellets are counted in a Wallac Wizard gamma counter.
Results are then analyzed and the dilution required to reach 50% of
the maximum binding value is calculated.
[0236] C. IC.sub.50 Determination for Peptide
[0237] To determine the IC.sub.50 of Peptide 1,100 .mu.L serial
dilutions of the peptide are combined with 100 .mu.L of recombinant
erythropoietin receptor (100 pg/tube) in 12.times.75 mm
polypropylene test tubes. Then 100 .mu.L of iodinated recombinant
human Erythropoietin (.sup.125I-EPO) is added to each tube and the
tubes are capped and mixed gently at 4.degree. C. overnight.
[0238] The next day, bound .sup.125I-EPO is quantitated as
described above. The results are analyzed and the IC.sub.50 value
calculated using Graphpad Prism version 4.0, from GraphPad
Software, Inc. (San Diego, Calif.). The assay is repeated two or
more times for each peptide tested, for a total of 3 or more
replicate IC.sub.50 determinations.
TABLE-US-00003 TABLE 2 In vitro activity assays for peptide dimers
Re- Pro- C/ Com- port- lifer- BFU- pound er ation e desig- EC50
EC50 IC50 EC90 nation Peptide dimer (pM) (nM) (pM) (nM) Pep- tide
II (SEQ ID NO: 3) ##STR00079## -- -- 110 2.2 Pep- tide III (SEQ ID
NO: 3) ##STR00080## 150 72 -- 2.7
Example 4
In Vivo Activity Assays
[0239] This example describes various in vivo assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. EPO-R agonist peptide dimers are
prepared according to the methods provided in Example 1 or Example
2. The in vivo activity of these peptide monomers and dimers is
evaluated using a series assays, including a polycythemic exhypoxic
mouse bioassay and a reticulocyte assay. These two assays are
described in further detail below.
1. Polycythemic Exhypoxic Mouse Bioassay
[0240] Test peptides are assayed for in vivo activity in the
polycythemic exhypoxic mouse bioassay adapted from the method
described by Cotes and Bangham (1961), Nature 191: 1065-1067. This
assay examines the ability of a test peptide to function as an EPO
mimetic: i.e., to activate EPO-R and induce new red blood cell
synthesis. Red blood cell synthesis is quantitated based upon
incorporation of radiolabeled iron into hemoglobin of the
synthesized red blood cells.
[0241] BDF1 mice are allowed to acclimate to ambient conditions for
7-10 days. Body weights are determined for all animals, and low
weight animals (<15 grams) are not used. Mice are subjected to
successive conditioning cycles in a hypobaric chamber for a total
of 14 days. Each 24 hour cycle consists of 18 hr at 0.40.+-.10.02%
atmospheric pressure and 6 hr at ambient pressure. After
conditioning, the mice are maintained at ambient pressure for an
additional 72 hr prior to dosing.
[0242] Test peptides, or recombinant human EPO standards, are
diluted in PBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock
solutions are first solubilized in dimethyl sulfoxide (DMSO).
Negative control groups include one group of mice injected with
PBS/BSA alone, and one group injected with 1% DMSO. Each dose group
contains 10 mice. Mice are injected subcutaneously (scruff of neck)
with 0.5 mL of the appropriate sample.
[0243] Forty eight hours following sample injection, the mice are
administered an intraperitoneal injection of 0.2 ml of Fe.sup.59
(Dupont, NEN), for a dose of approximately 0.75 .mu.Curies/mouse.
Mouse body weights are determined 24 hr after Fe.sup.59
administration, and the mice are sacrificed 48 hr after Fe.sup.59
administration. Blood is collected from each animal by cardiac
puncture and hematocrits are determined (heparin was used as the
anticoagulant). Each blood sample (0.2 ml) is analyzed for
Fe.sup.59 incorporation using a Packard gamma counter.
Non-responder mice (i.e., those mice with radioactive incorporation
less than the negative control group) are eliminated from the
appropriate data set. Mice that have hematocrit values less than
53% of the negative control group are also eliminated.
[0244] Results are derived from sets of 10 animals for each
experimental dose. The average amount of radioactivity incorporated
[counts per minute (CPM)] into blood samples from each group is
calculated.
2. Reticulocyte Assay
[0245] Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously)
on three consecutive days with either EPO control or test peptide.
At day three, mice are also dosed (0.1 mL, injected
intraperitoneally) with iron dextran (100 mg/ml). At day five, mice
are anesthetized with CO.sub.2 and bled by cardiac puncture. The
percent (%) reticulocytes for each blood sample is determined by
thiazole orange staining and flow cytometer analysis (retic-count
program). Hematocrits are manually determined. The corrected
percent of reticulocytes is determined using the following
formula:
% RETIC.sub.CORRECTED=%
RETIC.sub.OBSERVED.times.(Hematocrit.sub.INDIVIDUAL/Hematocrit.sub.NORMAL-
)
3. Hematological Assay
[0246] Normal CD1 mice are dosed with four weekly bolus intravenous
injections of either EPO positive control, test peptide, or
vehicle. A range of positive control and test peptide doses,
expressed as mg/kg, are tested by varying the active compound
concentration in the formulation. Volumes injected are 5 ml/kg. The
vehicle control group is comprised twelve animals, while 8 animals
are in each of the remaining dose groups. Daily viability and
weekly body weights are recorded.
[0247] The dosed mice are mice are fasted and then anesthetized
with inhaled isoflurane and terminal blood samples are collected
via cardiac or abdominal aorta puncture on Day 1 (for vehicle
control mice) and on Days 15 and 29 (4 mice/group/day). The blood
is transferred to Vacutainer.RTM. brand tubes. Preferred
anticoagulant is ethylenediaminetetraacetic acid (EDTA).
[0248] Blood samples are evaluated for endpoints measuring red
blood synthesis and physiology such as hematocrit (Hct), hemoglobin
(Hgb) and total erythrocyte count (RBC) using automated clinical
analyzers well known in the art (e.g., those made by Coulter,
Inc.).
Example 5
Synthesis of EPO-R Agonist Peptide Homodimers of Peptide Monomers
Having the Amino Acid Sequence (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ
ID NO: 1)
[0249] Step 1--Synthesis of Peptide Monomers:
[0250] Peptide monomers are synthesized using standard Fmoc
chemistry on an ABI 431A peptide synthesizer, using TO-RAM resin
(0.18 mmol/g Rapp Polymere, Germany). For the synthesis of peptide
monomers with an amidated carboxy terminus, the fully assembled
peptide is cleaved from the resin with 82.5% TFA, 5% water, 6.25%
anisole, 6.25% ethanedithiol. The deprotected product is filtered
from the resin and precipitated with diethyl ether. After thorough
drying, the product is purified by C18 reverse phase high
performance liquid chromatography with a gradient of
acetonitrile/water in 0.1% trifluoroacetic acid. The structure of
the peptide is confirmed by electrospray mass spectrometry. The
peptide is dissolved in a 1:1 solution of DMSO:water at a
concentration of 1 mg/mL to affect disulfide formation. The product
is purified by C18 reverse phase high performance liquid
chromatography with a gradient of acetonitrile/water in 0.1%
trifluoroacetic acid. The peptide monomers may be illustrated as
follows:
##STR00081##
[0251] Step 2--Synthesis of the Trifunctional Linker:
[0252] To a solution of diethyl iminoacetate (10.0 g, 52.8 mmol)
and Boc-beta-alanine (10.0 g, 52.8 mmol) in 100 mL of DCM was added
diisopropylcarbodiimide (8.0 mL, 51.1 mmol) over 10 minutes at room
temperature. The reaction mixture warmed to .about.10 degrees
during the addition, then cooled back to room temperature over 20
minutes. The reaction mixture was allowed to stir overnight and the
precipitated diisopropylurea was filtered off. The solvent was
removed under reduced pressure to afford a gum, and the residue
dissolved in ethyl acetate and again filtered to remove the
additional precipitated urea. The organic phase was placed into a
separatory funnel, washed (sat. NaHCO.sub.3, brine, 0.5 N HCl,
brine), dried (MgSO.sub.4), filtered and concentrated under reduced
pressure to afford the diester product as a colorless oil. The
diester was taken up in a 1:1 mixture of MeOH:THF (100 mL) and to
this was added water (25 mL), and then NaOH (5 g, 125 mmol). The pH
was measured to be >10. The reaction mixture was stirred at room
temperature for 2 h, and then acidified to pH 1 with 6N HCl. The
aq. Phase was saturated with NaCl and extracted 4 times with ethyl
acetate. The combined organic phase was washed (brine), dried
(MgSO.sub.4), and concentrated under reduced pressure to give a
white semi-solid. The solid was dissolved in 50 mL of DCM and to
this was added 300 mL hexane to create a white slurry. The solvent
was removed under reduced pressure to afford the diacid as a white
solid (14.7 g, 91.5% yield for 2 steps). To a solution of the
diacid (1 g, 3.29 mmol) in 20 mL of DMF was added
N-hydroxysuccinimide (770 mg, 6.69 mmol) and
diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and
4-dimethylaminopyridine (3 mg, 0.02 mmol). The reaction mixture was
stirred overnight and the solvent removed under reduced pressure.
The residue was taken up in ethyl acetate and filtered to remove
the precipitated urea. The organic phase was placed into a
separatory funnel, washed (sat. NaHCO.sub.3, brine, 0.5 N HCl,
brine), dried (MgSO.sub.4), filtered and concentrated under reduced
pressure to afford the di-NHS ester product as a white solid (1.12
g, 68% yield).
##STR00082##
[0253] Step 3--Coupling of the Trifunctional Linker to the Peptide
Monomers:
[0254] For coupling to the linker, 2 eq peptide is mixed with 1 eq
of trifunctional linker in dry DMF to give clear solution, 5 eq of
DIEA is added after 2 minutes. The mixture is stirred at ambient
temperature for 14 h. The solvent is removed under reduced pressure
and the crude product is dissolved in 80% TFA in DCM for 30 min to
remove the Boc group, followed by purification with C18 reverse
phase HPLC. The structure of the dimer is confirmed by electrospray
mass spectrometry. This coupling reaction attaches the linker to
the nitrogen atom of the .epsilon.-amino group of the lysine
residue of each monomer. The process is shown below with SEQ ID NO:
1.
##STR00083## ##STR00084##
[0255] Step 4--PEGylation of the Peptide Dimer:
[0256] PEGylation via a Carbamate Bond:
[0257] The peptide dimer is mixed with an equal amount (mole basis)
of activated PEG species (mPEG-NPC from NOF Corp. Japan) in dry DMF
to afford a clear solution. After minutes 4 eq of DIEA is added to
above solution. The mixture is stirred at ambient temperature 14 h,
followed by purification with C18 reverse phase HPLC. The structure
of PEGylated peptide is confirmed by MALDI mass. The purified
peptide was also subjected to purification via cation ion exchange
chromatography as outlined below. The mPEG-NPC PEGylation is shown
below using SEQ ID NO: 1.
##STR00085##
PEGylation Via an Amide Bond:
[0258] The peptide dimer is mixed with an equal amount (mole basis)
of activated PEG species (PEG-SPA-NHS from Shearwater Corp, USA) in
dry DMF to afford a clear solution. After 5 minutes 10 eq of DIEA
is added to above solution. The mixture is stirred at ambient
temperature 2 h, followed by purification with C18 reverse phase
HPLC. The structure of PEGylated peptide was confirmed by MALDI
mass. The purified peptide was also subjected to purification via
cation ion exchange chromatography as outlined below. The
PEG-SPA-NHS PEGylation is shown below using SEQ ID NO: 1.
##STR00086##
[0259] Step 5:--Ion exchange Purification of Peptides:
[0260] Several exchange supports were surveyed for their ability to
separate the above peptide-PEG conjugate from unreacted (or
hydrolyzed) PEG, in addition to their ability to retain the
starting dimeric peptides. The ion exchange resin (2-3 g) was
loaded into a 1 cm column, followed by conversion to the sodium
form (0.2 N NaOH loaded onto column until elutant was pH 14, ca. 5
column volumes), and than to the hydrogen form (eluted with either
0.1 N HCl or 0.1 M HOAc until elutant matched load pH, ca. 5 column
volumes), followed by washing with 25% ACN/water until pH 6. Either
the peptide prior to conjugation or the peptide-PEG conjugate was
dissolved in 25% ACN/water (10 mg/mL) and the pH adjusted to <3
with TFA, then loaded on the column. After washing with 2-3 column
volumes of 25% ACN/water and collecting 5 mL fractions, the peptide
was released from the column by elution with 0.1 M NH.sub.4OAc in
25% ACN/water, again collecting 5 mL fractions. Analysis via HPLC
revealed which fractions contained the desired peptide. Analysis
with an Evaporative Light-Scattering Detector (ELSD) indicated that
when the peptide was retained on the column and was eluted with the
NH.sub.4OAc solution (generally between fractions 4 and 10), no
non-conjugated PEG was observed as a contaminant. When the peptide
eluted in the initial wash buffer (generally the first 2
fractions), no separation of desired PEG-conjugate and excess PEG
was observed.
[0261] The following columns successfully retained both the peptide
and the peptide-PEG conjugate, and successfully purified the
peptide-PEG conjugate from the unconjugated peptide:
TABLE-US-00004 TABLE 3 Ion Exchange Resins Support Source Mono S HR
5/5 strong cation Amersham Biosciences exchange pre-loaded column
SE53 Cellulose, microgranular Whatman strong cation exchange
support SP Sepharose Fast Flow strong Amersham Biosciences cation
exchange support
Example 6
Synthesis of EPO-R Agonist Peptide Homodimers of Peptide Monomers
Having the Amino Acid Sequence (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K
(SEQ ID NO: 2)
[0262] EPO-R agonist peptide homodimers of peptide monomers having
the amino acid sequence (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID
NO: 2) are synthesized as described in Example 1, except that in
Step 1 the synthesized peptide monomers are:
TABLE-US-00005 (SEQ ID NO: 2)
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K
[0263] Where the PEG is attached to the linker via a carbamate
linkage, the final product of this synthesis using SEQ ID NO: 2 may
be illustrated structurally as follows:
##STR00087##
[0264] Where the PEG is attached to the linker via an amide
linkage, the final product of this synthesis using SEQ ID NO: 2 may
be illustrated structurally as follows:
##STR00088##
Example 7
In Vitro Activity Assays
[0265] This example describes various in vitro assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. The results for these assays demonstrate
that the novel peptides of this invention bind to EPO-R and
activate EPO-R signaling. Moreover, the results for these assays
show that the novel peptide compositions exhibit a surprising
increase in EPO-R binding affinity and biological activity compared
to EPO mimetic peptides that have been previously described.
[0266] EPO-R agonist peptide monomers and dimers are prepared
according to the methods provided in Example 1 or Example 2. The
potency of these peptide dimers is evaluated using a series of in
vitro activity assays, including: a reporter assay, a proliferation
assay, a competitive binding assay, and a C/BFU-e assay. These four
assays are described in further detail below.
[0267] The results of these in vitro activity assays are summarized
in Table 2.
1. Reporter Assay
[0268] This assay is based upon a murine pre-B-cell line derived
reporter cell, Baf3/EpoR/GCSFR fos/lux. This reporter cell line
expresses a chimeric receptor comprising the extra-cellular portion
of the human EPO receptor to the intra-cellular portion of the
human GCSF receptor. This cell line is further transfected with a
fos promoter-driven luciferase reporter gene construct. Activation
of this chimeric receptor through addition of erythropoietic agent
results in the expression of the luciferase reporter gene, and
therefore the production of light upon addition of the luciferase
substrate luciferin. Thus, the level of EPO-R activation in such
cells may be quantitated via measurement of luciferase
activity.
[0269] The Baf3/EpoR/GCSFR fos/lux cells are cultured in DMEM/F12
medium (Gibco) supplemented with 10% fetal bovine serum (FBS;
Hyclone), 10% WEHI-3 supernatant (the supernatant from a culture of
WEHI-3 cells, ATCC # TIB-68), and penicillin/streptomycin.
Approximately 18 h before the assay, cells are starved by
transferring them to DMEM/F12 medium supplemented with 10% FBS and
0.1% WEHI-3 supernatant. On the day of assay, cells are washed once
with DMEM/F12 medium supplemented with 10% FBS (no WEHI-3
supernatant), then 1.times.10.sup.6 cells/mL are cultured in the
presence of a known concentration of test peptide, or with EPO(R
& D Systems Inc., Minneapolis, Minn.) as a positive control, in
DMEM/F12 medium supplemented with 10% FBS (no WEHI-3 supernatant).
Serial dilutions of the test peptide are concurrently tested in
this assay. Assay plates are incubated for 4 h at 37.degree. C. in
a 5% CO.sub.2 atmosphere, after which luciferin (Steady-Glo;
Promega, Madison, Wis.) is added to each well. Following a 5-minute
incubation, light emission is measured on a Packard Topcount
Luminometer (Packard Instrument Co., Downers Grove, Ill.). Light
counts are plotted relative to test peptide concentration and
analysed using Graph Pad software. The concentration of test
peptide that results in a half-maximal emission of light is
recorded as the EC50
2. Proliferation Assay
[0270] This assay is based upon a murine pre-B-cell line, Baf3,
transfected to express human EPO-R. Proliferation of the resulting
cell line, BaF3/Ga14/Elk/EPOR, is dependent on EPO-R activation.
The degree of cell proliferation is quantitated using MTT, where
the signal in the MTT assay is proportional to the number of viable
cells.
[0271] The BaF3/Ga14/Elk/EPOR cells are cultured in spinner flasks
in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and
2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells are starved
overnight, in a spinner flask at a cell density of 1.times.10.sup.6
cells/ml, in DMEM/F12 medium supplemented with 10% FBS and 0.1%
WEHI-3 supernatant. The starved cells are then washed twice with
Dulbecco's PBS (Gibco), and resuspended to a density of
1.times.10.sup.6 cells/ml in DMEM/F12 supplemented with 10% FBS (no
WEHI-3 supernatant). 50 .mu.L aliquots (.about.50,000 cells) of the
cell suspension are then plated, in triplicate, in 96 well assay
plates. 50 .mu.L aliquots of dilution series of test EPO mimetic
peptides, or 504 EPO(R & D Systems Inc., Minneapolis, Minn.) or
Aranesp.TM. (darbepoeitin alpha, an ERO-R agonist commercially
available from Amgen) in DMEM/F12 media supplemented with 10% FBS
(no WEHI-3 supernatant I) are added to the 96 well assay plates
(final well volume of 100 .mu.L). For example, 12 different
dilutions may be tested where the final concentration of test
peptide (or control EPO peptide) ranges from 810 pM to 0.0045 pM.
The plated cells are then incubated for 48 h at 37.degree. C. Next,
10 .mu.L of MTT (Roche Diagnostics) is added to each culture dish
well, and then allowed to incubate for 4 h. The reaction is then
stopped by adding 10% SDS+0.01N HCl. The plates are then incubated
overnight at 37.degree. C. Absorbance of each well at a wavelength
of 595 nm is then measured by spectrophotometry. Plots of the
absorbance readings versus test peptide concentration are
constructed and the EC50 calculated using Graph Pad software. The
concentration of test peptide that results in a half-maximal
absorbance is recorded as the EC50.
3. Competitive Binding Assay
[0272] Competitive binding calculations are made using an assay in
which a light signal is generated as a function of the proximity of
two beads: a streptavidin donor bead bearing a biotinylated
EPO-R-binding peptide tracer and an acceptor bead to which is bound
EPO-R. Light is generated by non-radiative energy transfer, during
which a singlet oxygen is released from a first bead upon
illumination, and contact with the released singlet oxygen causes
the second bead to emit light. These bead sets are commercially
available (Packard). Bead proximity is generated by the binding of
the EPO-R-binding peptide tracer to the EPO-R. A test peptide that
competes with the EPO-R-binding peptide tracer for binding to EPO-R
will prevent this binding, causing a decrease in light
emission.
[0273] In more detail the method is as follows: Add 4 .mu.L of
serial dilutions of the test EPO-R agonist peptide, or positive or
negative controls, to wells of a 384 well plate. Thereafter, add 24
.mu.L/well of receptor/bead cocktail. Receptor bead cocktail
consists of: 15 .mu.L of 5 mg/ml streptavidin donor beads
(Packard), 15 .mu.L of 5 mg/ml monoclonal antibody ab179 (this
antibody recognizes the portion of the human placental alkaline
phosphatase protein contained in the recombinant EPO-R), protein
A-coated acceptor beads (protein A will bind to the ab179 antibody;
Packard), 112.5 .mu.L of a 1:6.6 dilution of recombinant EPO-R
(produced in Chinese Hamster Ovary cells as a fusion protein to a
portion of the human placental alkaline phosphatase protein which
contains the ab179 target epitope) and 607.5 .mu.L of Alphaquest
buffer (40 mM HEPES, pH 7.4; 1 mM MgCl.sub.2; 0.1% BSA, 0.05% Tween
20). Tap to mix. Add 2 .mu.L/well of the biotinylated EPO-R-binding
peptide tracer, (30 nM final concentration). The petpide tracer, an
EPO-R binding peptide (see in the tables "Reporter EC50 (pM)"), is
made according to the methods described in Example 1, using SEQ ID
NO: 4.
##STR00089##
[0274] Centrifuge 1 min to mix. Seal plate with Packard Top Seal
and wrap in foil. Incubate overnight at room temperature. After 18
hours read light emission using an AlphaQuest reader (Packard).
Plot light emission vs. concentration of peptide and analyze with
Graph Pad or Excel.
[0275] The concentration of test peptide that results in a 50%
decrease in light emission, relative to that observed without test
peptide, is recorded as the IC50.
4. C/BFU-e Assay
[0276] EPO-R signaling stimulates the differentiation of bone
marrow stem cells into proliferating red blood cell precursors.
This assay measures the ability of test peptides to stimulate the
proliferation and differentiation of red blood cell precursors from
primary human bone marrow pluripotent stem cells.
[0277] For this assay, serial dilutions of test peptide are made in
IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These
serial dilutions, or positive control EPO peptide, are then added
to methylcellulose to give a final volume of 1.5 mL. The
methylcellulose and peptide mixture is then vortexed thoroughly.
Aliquots (100,000 cells/mL) of human, bone marrow derived CD34+
cells (Poietics/Cambrex) are thawed. The thawed cells are gently
added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50 mL tube.
Next, 40-50 mL IMDM medium is added gently to cells: the medium is
added drop by drop along the side of the 50 mL tube for the first
10 mL, and then the remaining volume of medium is slowly dispensed
along the side of the tube. The cells are then spun at 900 rpm for
20 min, and the media removed carefully by gentle aspiration. The
cells are resuspended in 1 ml of IMDM medium and the cell density
per mL is counted on hemacytometer slide (10 .mu.L aliquot of cell
suspension on slide, and cell density is the average count X 10,000
cells/ml). The cells are then diluted in IMDM medium to a cell
density of 15,000 cells/mL. A 100 .mu.L of diluted cells is then
added to each 1.5 mL methyl cellulose plus peptide sample (final
cell concentration in assay media is 1000 cells/mL), and the
mixture is vortexed. Allow the bubbles in the mixture to disappear,
and then aspirate 1 mL using blunt-end needle. Add 0.25 mL
aspirated mixture from each sample into each of 4 wells of a
24-well plate (Falcon brand). Incubate the plated mixtures at
37.degree. C. under 5% CO.sub.2 in a humid incubator for 14 days.
Score for the presence of erythroid colonies using a phase
microscope (5.times.-10.times. objective, final magnification of
100.times.). The concentration of test peptide at which the number
of formed colonies is 90% of maximum, relative to that observed
with the EPO positive control, is recorded as the EC90 [See Table
2: C/BFU-e EC90].
TABLE-US-00006 TABLE 4 In vitro activity assays for peptide dimers
Re- Pro- C/ Com- port- lifer- BFU- pound er ation AQ e desig- EC50
EC50 IC50 EC90 nation Peptide dimer (pM) (pM) (pM) (nM) Pep- tide
IV (SEQ ID NO: 1) ##STR00090## -- -- -- 6.2
Example 8
In Vivo Activity Assays
[0278] This example describes various in vivo assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. EPO-R agonist peptide monomers and
dimers are prepared according to the methods provided in Example 1.
The in vivo activity of these peptide monomers and dimers is
evaluated using a series assays, including a polycythemic exhypoxic
mouse bioassay and a reticulocyte assay. These two assays are
described in further detail below.
1. Polycythemic Exhypoxic Mouse Bioassay
[0279] Test peptides are assayed for in vivo activity in the
polycythemic exhypoxic mouse bioassay adapted from the method
described by Cotes and Bangham (1961), Nature 191: 1065-1067. This
assay examines the ability of a test peptide to function as an EPO
mimetic: i.e., to activate EPO-R and induce new red blood cell
synthesis. Red blood cell synthesis is quantitated based upon
incorporation of radiolabeled iron into hemoglobin of the
synthesized red blood cells.
[0280] BDF1 mice are allowed to acclimate to ambient conditions for
7-10 days. Body weights are determined for all animals, and low
weight animals (<15 grams) are not used. Mice are subjected to
successive conditioning cycles in a hypobaric chamber for a total
of 14 days. Each 24 hour cycle consists of 18 hr at 0.40.+-.0.02%
atmospheric pressure and 6 hr at ambient pressure. After
conditioning the mice are maintained at ambient pressure for an
additional 72 hr prior to dosing.
[0281] Test peptides, or recombinant human EPO standards, are
diluted in PBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock
solutions are first solubilized in dimethyl sulfoxide (DMSO).
Negative control groups include one group of mice injected with
PBS/BSA alone, and one group injected with 1% DMSO. Each dose group
contains 10 mice. Mice are injected subcutaneously (scruff of neck)
with 0.5 mL of the appropriate sample.
[0282] Forty eight hours following sample injection, the mice are
administered an intraperitoneal injection of 0.2 ml of Fe.sup.59
(Dupont, NEN), for a dose of approximately 0.75 .mu.Curies/mouse.
Mouse body weights are determined 24 hr after Fe.sup.59
administration, and the mice are sacrificed 48 hr after Fe.sup.59
administration. Blood is collected from each animal by cardiac
puncture and hematocrits are determined (heparin was used as the
anticoagulant). Each blood sample (0.2 ml) is analyzed for
Fe.sup.59 incorporation using a Packard gamma counter.
Non-responder mice (i.e., those mice with radioactive incorporation
less than the negative control group) are eliminated from the
appropriate data set. Mice that have hematocrit values less than
53% of the negative control group are also eliminated.
[0283] Results are derived from sets of 10 animals for each
experimental dose. The average amount of radioactivity incorporated
[counts per minute (CPM)] into blood samples from each group is
calculated.
2. Reticulocyte Assay
[0284] Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously)
on three consecutive days with either EPO control or test peptide.
At day three, mice are also dosed (0.1 mL, injected
intraperitoneally) with iron dextran (100 mg/ml). At day five, mice
are anesthetized with CO.sub.2 and bled by cardiac puncture. The
percent (%) reticulocytes for each blood sample is determined by
thiazole orange staining and flow cytometer analysis (retic-count
program). Hematocrits are manually determined. The corrected
percent of reticulocytes is determined using the following
formula:
% RETIC.sub.CORRECTED=%
RETIC.sub.OBSERVED.times.(Hematocrit.sub.INDIVIDUAL/Hematocrit.sub.NORMAL-
)
3. Hematological Assay
[0285] Normal CD1 mice are dosed with four weekly bolus intravenous
injections of either EPO positive control, test peptide, or
vehicle. A range of positive control and test peptide doses,
expressed as mg/kg, are tested by varying the active compound
concentration in the formulation. Volumes injected are 5 ml/kg. The
vehicle control group is comprised twelve animals, while 8 animals
are in each of the remaining dose groups. Daily viability and
weekly body weights are recorded.
[0286] The dosed mice are mice are fasted and then anesthetized
with inhaled isoflurane and terminal blood samples are collected
via cardiac or abdominal aorta puncture on Day 1 (for vehicle
control mice) and on Days 15 and 29 (4 mice/group/day). The blood
is transferred to Vacutainer.RTM. brand tubes. Preferred
anticoagulant is ethylenediaminetetraacetic acid (EDTA).
[0287] Blood samples are evaluated for endpoints measuring red
blood synthesis and physiology such as hematocrit (Hct), hemoglobin
(Hgb) and total erythrocyte count (RBC) using automated clinical
analyzers well known in the art (e.g., those made by Coulter,
Inc.).
Example 9
Synthesis of EPO-R Agonist Peptide Homodimers of Peptide Monomers
Having the Amino Acid Sequence (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ
ID NO: 1)
[0288] Step 1--Synthesis of Peptide Monomers:
[0289] Peptide monomers are synthesized using standard Fmoc
chemistry on an ABI 431A peptide synthesizer, using TG-RAM resin
(0.18 mmol/g Rapp Polymere, Germany). For the synthesis of peptide
monomers with an amidated carboxy terminus, the fully assembled
peptide is cleaved from the resin with 82.5% TFA, 5% water, 6.25%
anisole, 6.25% ethanedithiol. The deprotected product is filtered
from the resin and precipitated with diethyl ether. After thorough
drying the product is purified by C18 reverse phase high
performance liquid chromatography with a gradient of
acetonitrile/water in 0.1% trifluoroacetic acid. The structure of
the peptide is confirmed by electrospray mass spectrometry. The
peptide monomers may be illustrated as follows:
TABLE-US-00007 (SEQ ID NO: 1)
(AcG)GLYACHMGPIT(1-nal)VCQPLRK-NH.sub.2
[0290] Step 2--Synthesis of the Trifunctional Linker:
[0291] To a solution of diethyl iminoacetate (10.0 g, 52.8 mmol)
and Boc-beta-alanine (10.0 g, 52.8 mmol) in 100 mL of DCM was added
diisopropylcarbodiimide (8.0 mL, 51.1 mmol) over 10 minutes at room
temperature. The reaction mixture warmed to .about.10 degrees
during the addition, then cooled back to room temperature over 20
minutes. The reaction mixture was allowed to stir overnight and the
precipitated diisopropylurea was filtered off. The solvent was
removed under reduced pressure to afford a gum, and the residue
dissolved in ethyl acetate and again filtered to remove the
additional precipitated urea. The organic phase was placed into a
separatory funnel, washed (sat. NaHCO.sub.3, brine, 0.5 N HCl,
brine), dried (MgSO.sub.4), filtered and concentrated under reduced
pressure to afford the diester product as a colorless oil. The
diester was taken up in a 1:1 mixture of MeOH:THF (100 mL) and to
this was added water (25 mL), and then NaOH (5 g, 125 mmol). The pH
was measured to be >10. The reaction mixture was stirred at room
temperature for 2 h, and then acidified to pH 1 with 6N HCl. The
aq. Phase was saturated with NaCl and extracted 4 times with ethyl
acetate. The combined organic phase was washed (brine), dried
(MgSO.sub.4), and concentrated under reduced pressure to give a
white semi-solid. The solid was dissolved in 50 mL of DCM and to
this was added 300 mL hexane to create a white slurry. The solvent
was removed under reduced pressure to afford the diacid as a white
solid (14.7 g, 91.5% yield for 2 steps). To a solution of the
diacid (1 g, 3.29 mmol) in 20 mL of DMF was added
N-hydroxysuccinimide (770 mg, 6.69 mmol) and
diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and
4-dimethylaminopyridine (3 mg, 0.02 mmol). The reaction mixture was
stirred overnight and the solvent removed under reduced pressure.
The residue was taken up in ethyl acetate and filtered to remove
the precipitated urea. The organic phase was placed into a
separatory funnel, washed (sat. NaHCO.sub.3, brine, 0.5 N HCl,
brine), dried (MgSO.sub.4), filtered and concentrated under reduced
pressure to afford the di-NHS ester product as a white solid (1.12
g, 68% yield).
##STR00091##
[0292] Step 3--Coupling of The Trifunctional Linker to The Peptide
Monomers:
[0293] For coupling to the linker, 2 eq peptide is mixed with 1 eq
of trifunctional linker in dry DMF to give a clear solution, and 5
eq of DIEA is added after 2 minutes. The mixture is stirred at
ambient temperature for 14 h. The solvent is removed under reduced
pressure and the crude product is dissolved in 80% TFA in DCM for
30 min to remove the Boc group, followed by purification with C18
reverse phase HPLC. The structure of the dimer is confirmed by
electrospray mass spectrometry. This coupling reaction attaches the
linker to the nitrogen atom of the .epsilon.-amino group of the
lysine residue of each monomer. Coupling using SEQ ID NO: 1 is
shown below.
##STR00092## ##STR00093##
[0294] Step 4--Synthesis of PEG Moiety Comprising Two Linear PEG
Chains Linked by Lysine mPEG2-Lysinol-NPC
[0295] Lysinol, which may be obtained commercially, is treated with
an excess of mPEG2-NPC to obtain MPEG2-lysinol, which is then
reacted with NPC to form mPEG2-lysinol-NPC.
mPEG2-Lys-NHS
[0296] This product may be obtained commercially, for example, from
the Molecular Engineering catalog (2003) of Nektar Therapeutics
(490 Discovery Drive, Huntsville, Ala. 35806), item no.
2Z3X0T01.
[0297] Step 5--PEGylation of the Peptide Dimer:
PEGylation Via a Carbamate Bond:
[0298] The peptide dimer and the PEG species
(mPEG.sub.2-Lysinol-NPC) are mixed in a 1:2 molar ratio in dry DMF
to afford a clear solution. After 5 minutes 4 eq of DIEA is added
to above solution. The mixture is stirred at ambient temperature 14
h, followed by purification with C18 reverse phase HPLC. The
structure of PEGylated peptide is confirmed by MALDI mass. The
purified peptide was also subjected to purification via cation ion
exchange chromatography as outlined below. PEGylation using
mPEG-Lysinol-NPC is shown below using SEQ ID NO: 1.
##STR00094##
PEGylation Via an Amide Bond:
[0299] The peptide dimer and PEG species (mPEG.sub.2-Lys-NHS from
Shearwater Corp, USA) are mixed in a 1:2 molar ratio in dry DMF to
afford a clear solution. After 5 minutes 10 eq of DIEA is added to
above solution. The mixture is stirred at ambient temperature 2 h,
followed by purification with C18 reverse phase HPLC. The structure
of PEGylated peptide was confirmed by MALDI mass. The purified
peptide was also subjected to purification via cation ion exchange
chromatography as outlined below. PEGylation using
mPEG.sub.2-Lys-NHS using SEQ ID NO: 1 is shown below.
##STR00095##
[0300] Step 6:--Ion Exchange Purification of Peptides:
[0301] Several exchange supports were surveyed for their ability to
separate the above peptide-PEG conjugate from unreacted (or
hydrolyzed) PEG, in addition to their ability to retain the
starting dimeric peptides. The ion exchange resin (2-3 g) was
loaded into a 1 cm column, followed by conversion to the sodium
form (0.2 N NaOH loaded onto column until elutant was pH 14, ca. 5
column volumes), and than to the hydrogen form (eluted with either
0.1 N HCl or 0.1 M HOAc until elutant matched load pH, ca. 5 column
volumes), followed by washing with 25% ACN/water until pH 6. Either
the peptide prior to conjugation or the peptide-PEG conjugate was
dissolved in 25% ACN/water (10 mg/mL) and the pH adjusted to <3
with TFA, then loaded on the column. After washing with 2-3 column
volumes of 25% ACN/water and collecting 5 mL fractions, the peptide
was released from the column by elution with 0.1 M NH.sub.4OAc in
25% ACN/water, again collecting 5 mL fractions. Analysis via HPLC
revealed which fractions contained the desired peptide. Analysis
with an Evaporative Light-Scattering Detector (ELSD) indicated that
when the peptide was retained on the column and was eluted with the
NH.sub.4OAc solution (generally between fractions 4 and 10), no
non-conjugated PEG was observed as a contaminant. When the peptide
eluted in the initial wash buffer (generally the first 2
fractions), no separation of desired PEG-conjugate and excess PEG
was observed.
[0302] The following columns successfully retained both the peptide
and the peptide-PEG conjugate, and successfully purified the
peptide-PEG conjugate from the unconjugated peptide:
TABLE-US-00008 TABLE 5 Ion Exchange Resins Support Source Mono S HR
5/5 strong cation Amersham Biosciences exchange pre-loaded column
SE53 Cellulose, microgranular Whatman strong cation exchange
support SP Sepharose Fast Flow strong Amersham Biosciences cation
exchange support
Example 10
Synthesis of EPO-R Agonist Peptide Homodimers of Peptide Monomers
Having the Amino Acid Sequence (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K
(SEQ ID NO: 2)
[0303] EPO-R agonist peptide homodimers of peptide monomers having
the amino acid sequence (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID
NO: 2) are synthesized as described in Example 1, except that in
Step 1 the synthesized peptide monomers are:
TABLE-US-00009 (SEQ ID NO: 2)
(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K
[0304] Where the PEG is attached to the spacer via carbamate
linkages, the final product of this synthesis using SEQ ID NO: 2
may be illustrated structurally as follows:
##STR00096##
[0305] Where the PEG is attached to the spacer via amide linkages,
the final product of this synthesis using SEQ ID NO: 2 may be
illustrated structurally as follows:
##STR00097##
Example 11
In Vitro Activity Assays
[0306] This example describes various in vitro assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. The results for these assays demonstrate
that the novel peptides of this invention bind to EPO-R and
activate EPO-R signaling. Moreover, the results for these assays
show that the novel peptide compositions exhibit a surprising
increase in EPO-R binding affinity and biological activity compared
to EPO mimetic peptides that have been previously described.
[0307] EPO-R agonist peptide monomers and dimers are prepared
according to the methods provided in Example 1 or Example 2. The
potency of these peptide dimers is evaluated using a series of in
vitro activity assays, including: a reporter assay, a proliferation
assay, a competitive binding assay, and a C/BFU-e assay. These four
assays are described in further detail below.
[0308] The results of these in vitro activity assays are summarized
in Table 2.
I. Reporter Assay
[0309] This assay is based upon a on a murine pre-B-cell line
derived reporter cell, Baf3/EpoR/GCSFR fos/lux. This reporter cell
line expresses a chimeric receptor comprising the extra-cellular
portion of the human EPO receptor to the intra-cellular portion of
the human GCSF receptor. This cell line is further transfected with
a fos promoter-driven luciferase reporter gene construct.
Activation of this chimeric receptor through addition of
erythropoietic agent results in the expression of the luciferase
reporter gene, and therefore the production of light upon addition
of the luciferase substrate luciferin. Thus, the level of EPO-R
activation in such cells may be quantitated via measurement of
luciferase activity.
[0310] The Baf3/EpoR/GCSFR fos/lux cells are cultured in DMEM/F12
medium (Gibco) supplemented with 10% fetal bovine serum (FBS;
Hyclone), 10% WEHI-3 supernatant (the supernatant from a culture of
WEHI-3 cells, ATCC # TIB-68), and penicillin/streptomycin.
Approximately 18 h before the assay, cells are starved by
transferring them to DMEM/F12 medium supplemented with 10% FBS and
0.1% WEHI-3 supernatant. On the day of assay, cells are washed once
with DMEM/F12 medium supplemented with 10% FBS (no WEHI-3
supernatant), then 1.times.10.sup.6 cells/mL are cultured in the
presence of a known concentration of test peptide, or with EPO(R
& D Systems Inc., Minneapolis, Minn.) as a positive control, in
DMEM/F12 medium supplemented with 10% FBS (no WEHI-3 supernatant).
Serial dilutions of the test peptide are concurrently tested in
this assay. Assay plates are incubated for 4 h at 37.degree. C. in
a 5% CO.sub.2 atmosphere, after which luciferin (Steady-Glo;
Promega, Madison, Wis.) is added to each well. Following a 5-minute
incubation, light emission is measured on a Packard Topcount
Luminometer (Packard Instrument Co., Downers Grove, Ill.). Light
counts are plotted relative to test peptide concentration and
analysed using Graph Pad software. The concentration of test
peptide that results in a half-maximal emission of light is
recorded as the EC50
2. Proliferation Assay
[0311] This assay is based upon a murine pre-B-cell line, Baf3,
transfected to express human EPO-R. Proliferation of the resulting
cell line, BaF3/Ga14/Elk/EPOR, is dependent on EPO-R activation.
The degree of cell proliferation is quantitated using MTT, where
the signal in the MTT assay is proportional to the number of viable
cells.
[0312] The BaF3/Ga14/Elk/EPOR cells are cultured in spinner flasks
in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and
2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells are starved
overnight, in a spinner flask at a cell density of 1.times.10.sup.6
cells/ml, in DMEM/F12 medium supplemented with 10% FBS and 0.1%
WEHI-3 supernatant. The starved cells are then washed twice with
Dulbecco's PBS (Gibco), and resuspended to a density of
1.times.10.sup.6 cells/ml in DMEM/F12 supplemented with 10% FBS (no
WEHI-3 supernatant). 504 aliquots (.about.50,000 cells) of the cell
suspension are then plated, in triplicate, in 96 well assay plates.
504 aliquots of dilution series of test EPO mimetic peptides, or 50
.mu.L EPO(R & D Systems Inc., Minneapolis, Minn.) or
Aranesp.TM. (darbepoeitin alpha, an ERO-R agonist commercially
available from Amgen) in DMEM/F12 media supplemented with 10% FBS
(no WEHI-3 supernatant I) are added to the 96 well assay plates
(final well volume of 100 .mu.L). For example, 12 different
dilutions may be tested where the final concentration of test
peptide (or control EPO peptide) ranges from 810 pM to 0.0045 pM.
The plated cells are then incubated for 48 h at 37.degree. C. Next,
10 .mu.L of MTT (Roche Diagnostics) is added to each culture dish
well, and then allowed to incubate for 4 h. The reaction is then
stopped by adding 10% SDS+0.01N HCl. The plates are then incubated
overnight at 37.degree. C. Absorbance of each well at a wavelength
of 595 nm is then measured by spectrophotometry. Plots of the
absorbance readings versus test peptide concentration are
constructed and the EC50 calculated using Graph Pad software. The
concentration of test peptide that results in a half-maximal
absorbance is recorded as the EC50.
3. Competitive Binding Assay
[0313] Competitive binding calculations are made using an assay in
which a light signal is generated as a function of the proximity of
two beads: a streptavidin donor bead bearing a biotinylated
EPO-R-binding peptide tracer and an acceptor bead to which is bound
EPO-R. Light is generated by non-radiative energy transfer, during
which a singlet oxygen is released from a first bead upon
illumination, and contact with the released singlet oxygen causes
the second bead to emit light. These bead sets are commercially
available (Packard). Bead proximity is generated by the binding of
the EPO-R-binding peptide tracer to the EPO-R. A test peptide that
competes with the EPO-R-binding peptide tracer for binding to EPO-R
will prevent this binding, causing a decrease in light
emission.
[0314] In more detail the method is as follows: Add 4 .mu.L of
serial dilutions of the test EPO-R agonist peptide, or positive or
negative controls, to wells of a 384 well plate. Thereafter, add 2
.mu.L/well of receptor/bead cocktail. Receptor bead cocktail
consists of 15 .mu.L of 5 mg/ml streptavidin donor beads (Packard),
15 .mu.L of 5 mg/ml monoclonal antibody ab179 (this antibody
recognizes the portion of the human placental alkaline phosphatase
protein contained in the recombinant EPO-R), protein A-coated
acceptor beads (protein A will bind to the ab179 antibody;
Packard), 112.5 .mu.L of a 1:6.6 dilution of recombinant EPO-R
(produced in Chinese Hamster Ovary cells as a fusion protein to a
portion of the human placental alkaline phosphatase protein which
contains the ab179 target epitope) and 607.54 of Alphaquest buffer
(40 mM HEPES, pH 7.4; 1 mM MgCl.sub.2; 0.1% BSA, 0.05% Tween 20).
Tap to mix. Add 2 .mu.L/well of the biotinylated EPO-R-binding
peptide tracer, (30 nM final concentration). The peptide tracer, an
EPO-R binding peptide (see in the tables "Reporter EC50 (pM)"), is
made according to the methods described in Example 1 using SEQ ID
NO: 4.
##STR00098##
[0315] Centrifuge 1 min to mix. Seal plate with Packard Top Seal
and wrap in foil. Incubate overnight at room temperature. After 18
hours read light emission using an AlphaQuest reader (Packard).
Plot light emission vs. concentration of peptide and analyze with
Graph Pad or Excel.
[0316] The concentration of test peptide that results in a 50%
decrease in light emission, relative to that observed without test
peptide, is recorded as the IC50.
4. C/BFU-e Assay
[0317] EPO-R signaling stimulates the differentiation of bone
marrow stern cells into proliferating red blood cell precursors.
This assay measures the ability of test peptides to stimulate the
proliferation and differentiation of red blood cell precursors from
primary human bone marrow pluripotent stem cells.
[0318] For this assay, serial dilutions of test peptide are made in
IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These
serial dilutions, or positive control EPO peptide, are then added
to methylcellulose to give a final volume of 1.5 mL. The
methylcellulose and peptide mixture is then vortexed thoroughly.
Aliquots (100,000 cells/mL) of human, bone marrow derived CD34+
cells (Poietics/Cambrex) are thawed. The thawed cells are gently
added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50 mL tube.
Next, 40-50 mL IMDM medium is added gently to cells: the medium is
added drop by drop along the side of the 50 mL tube for the first
10 mL, and then the remaining volume of medium is slowly dispensed
along the side of the tube. The cells are then spun at 900 rpm for
20 min, and the media removed carefully by gentle aspiration. The
cells are resuspended in 1 ml of IMDM medium and the cell density
per mL is counted on hemacytometer slide (104 aliquot of cell
suspension on slide, and cell density is the average count X 10,000
cells/ml). The cells are then diluted in IMDM medium to a cell
density of 15,000 cells/mL. A 100 .mu.L of diluted cells is then
added to each 1.5 mL methyl cellulose plus peptide sample (final
cell concentration in assay media is 1000 cells/mL), and the
mixture is vortexed. Allow the bubbles in the mixture to disappear,
and then aspirate 1 mL using blunt-end needle. Add 0.25 mL
aspirated mixture from each sample into each of 4 wells of a
24-well plate (Falcon brand). Incubate the plated mixtures at
37.degree. C. under 5% CO.sub.2 in a humid incubator for 14 days.
Score for the presence of erythroid colonies using a phase
microscope (5.times.-10.times. objective, final magnification of
100.times.). The concentration of test peptide at which the number
of formed colonies is 90% of maximum, relative to that observed
with the EPO positive control, is recorded as the EC90 [See Table
2: C/BFU-e EC90].
TABLE-US-00010 TABLE 6 In vitro activity assays for peptide dimers
Re- Pro- Radio- C/ Com- port- lifer- li- BFU- pound er ation gand e
desig- EC50 EC50 IC50 EC90 nation Peptide dimer (pM) (pM) (pM) (nM)
Pep- tide I (SEQ ID NO: 2) ##STR00099## 195 165 111 3
Example 12
In Vivo Activity Assays
[0319] This example describes various in vivo assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. EPO-R agonist peptide monomers and
dimers are prepared according to the methods provided in Example 1.
The in vivo activity of these peptide monomers and dimers is
evaluated using a series assays, including a polycythemic exhypoxic
mouse bioassay and a reticulocyte assay. These two assays are
described in further detail below.
1. Polycythemic Exhypoxic Mouse Bioassay
[0320] Test peptides are assayed for in vivo activity in the
polycythemic exhypoxic mouse bioassay adapted from the method
described by Cotes and Bangham (1961), Nature 191: 1065-1067. This
assay examines the ability of a test peptide to function as an EPO
mimetic: to activate EPO-R and induce new red blood cell synthesis.
Red blood cell synthesis is quantitated based upon incorporation of
radiolabeled iron into hemoglobin of the synthesized red blood
cells.
[0321] BDF1 mice are allowed to acclimate to ambient conditions for
7-10 days. Body weights are determined for all animals, and low
weight animals (<15 grams) are not used. Mice are subjected to
successive conditioning cycles in a hypobaric chamber for a total
of 14 days. Each 24 hour cycle consists of 18 hr at 0.40.+-.0.02%
atmospheric pressure and 6 hr at ambient pressure. After
conditioning the mice are maintained at ambient pressure for an
additional 72 hr prior to dosing.
[0322] Test peptides, or recombinant human EPO standards, are
diluted in PBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock
solutions are first solubilized in dimethyl sulfoxide (DMSO).
Negative control groups include one group of mice injected with
PBS/BSA alone, and one group injected with 1% DMSO. Each dose group
contains 10 mice. Mice are injected subcutaneously (scruff of neck)
with 0.5 mL of the appropriate sample.
[0323] Forty eight hours following sample injection, the mice are
administered an intraperitoneal injection of 0.2 ml of Fe.sup.59
(Dupont, NEN), for a dose of approximately 0.75 .mu.Curies/mouse.
Mouse body weights are determined 24 hr after Fe.sup.59
administration, and the mice are sacrificed 48 hr after Fe.sup.59
administration. Blood is collected from each animal by cardiac
puncture and hematocrits are determined (heparin was used as the
anticoagulant). Each blood sample (0.2 ml) is analyzed for
Fe.sup.59 incorporation using a Packard gamma counter.
Non-responder mice (i.e., those mice with radioactive incorporation
less than the negative control group) are eliminated from the
appropriate data set. Mice that have hematocrit values less than
53% of the negative control group are also eliminated.
[0324] Results are derived from sets of 10 animals for each
experimental dose. The average amount of radioactivity incorporated
[counts per minute (CPM)] into blood samples from each group is
calculated.
2. Reticulocyte Assay
[0325] Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously)
on three consecutive days with either EPO control or test peptide.
At day three, mice are also dosed (0.1 mL, injected
intraperitoneally) with iron dextran (100 mg/ml). At day five, mice
are anesthetized with CO.sub.2 and bled by cardiac puncture. The
percent (%) reticulocytes for each blood sample is determined by
thiazole orange staining and flow cytometer analysis (retic-count
program). Hematocrits are manually determined. The corrected
percent of reticulocytes is determined using the following
formula:
% RETIC.sub.CORRECTED=%
RETIC.sub.OBSERVED.times.(Hematocrit.sub.INDIVIDUAL/Hematocrit.sub.NORMAL-
)
3. Hematological Assay
[0326] Normal CD1 mice are dosed with four weekly bolus intravenous
injections of either EPO positive control, test peptide, or
vehicle. A range of positive control and test peptide doses,
expressed as mg/kg, are tested by varying the active compound
concentration in the formulation. Volumes injected are 5 ml/kg. The
vehicle control group is comprised twelve animals, while 8 animals
are in each of the remaining dose groups. Daily viability and
weekly body weights are recorded.
[0327] The dosed mice are mice are fasted and then anesthetized
with inhaled isoflurane and terminal blood samples are collected
via cardiac or abdominal aorta puncture on Day 1 (for vehicle
control mice) and on Days 15 and 29 (4 mice/group/day). The blood
is transferred to Vacutainer.RTM. brand tubes. Preferred
anticoagulant is ethylenediaminetetraacetic acid (EDTA).
[0328] Blood samples are evaluated for endpoints measuring red
blood synthesis and physiology such as hematocrit (Hct), hemoglobin
(Hgb) and total erythrocyte count (RBC) using automated clinical
analyzers well known in the art (e.g., those made by Coulter,
Inc.).
Example 13
Increase in Hemoglobin Levels in Animals and Human Normal Healthy
Volunteers (NHV)
1. General
[0329] Based on nonclinical and clinical data, Peptide I, a fully
synthetic EPO receptor agonist, has the potential to safely and
effectively alleviate anemia secondary to inadequate EPO
production. It is anticipated that Peptide I may have several
potential advantages over currently available EPO products,
including: prolonged half-life and pharmacodynamic activity with an
expected dosing interval of every 3 to 4 weeks which may translate
into improved convenience and compliance; reduced potential for
antibody mediated pure red cell aplasia (PRCA) since there is no
common amino acid sequence shared with endogenous EPO; potential
for the treatment of patients with PRCA caused by antibodies to
commercially available EPOs that cross-react with endogenous EPO;
and enhanced stability with prolonged shelf life at room
temperature compared to protein therapeutics.
[0330] Since the primary amino acid sequence of Peptide I differs
from that of recombinant Human EPO (rHuEPO), it is less likely to
induce a cross-reactive immune response against endogenous EPO.
Although very rare, a cross-reactive immune response can cause
serious side effects associated with loss of potency for both
recombinant human ESAs and endogenous EPO. In addition, since
Peptide I is a synthetic peptide, its production avoids the
potential risk of contamination of the drug with host cell material
that may occur with recombinant protein products.
2. Pharmacokinetics of Peptide I in Animals
[0331] Peptide I is a potent stimulator of erythropoiesis with
dose-dependent activity observed following single or repeat dose
administration in mice, rats (normocythemic and nephrectomized),
dogs, rabbits and monkeys. Pharmacology studies in rats and monkeys
indicate that the rise in reticulocytes (immature RBCs) and RBCs as
measured by hemoglobin levels is dose proportional.
[0332] Pharmacokinetic studies in rats, dogs and monkeys have
demonstrated the sustained plasma persistence of Peptide I in
comparison to currently marketed rHuEPO products. Elimination half
life (t.sub.1/2) ranges from 21.5-30.7 hours in rats, to 73.7 hours
in dogs. Biodistribution of Peptide I is primarily to the plasma
fraction. Following IV administration of 9.87 mg Peptide I/kg to
five-sixth nephrectomy rats, the t.sub.1/2 was approximately 48
hours and clearance was low at 0.763 mL/h/kg (compared to 1.44
mL/h/kg in normocythemic rats) resulting in an increased AUC
magnitude of 1.8.
2. Pharmacodynamics and Pharmacokinetics of Peptide I in Humans
[0333] 2.1, Overview and Methods
[0334] Peptide I has been tested in 20 NHV in one Phase 1 study.
This first-in-human study with Peptide I was a randomized,
double-blind, placebo-controlled, single-dose, IV, escalating dose,
safety and tolerability trial. The primary objectives of the study
were to evaluate the safety and pharmacokinetics of Peptide I, and
to establish a pharmacologically active dose (PAD). Cohorts of
seven male volunteers were scheduled to receive single doses of
Peptide I or placebo in a 5:2 ratio. Cohorts were to be added at
increasing dose levels until a PAD, as determined by an increase in
hemoglobin from baseline values, was observed, at which point the
dose level identified as the PAD would be repeated in another
cohort of volunteers.
[0335] The study was initiated in four cohorts were enrolled in a
consecutive manner at Peptide I doses of 0.025, 0.05, 0.1 mg/kg,
and 0.1 mg/kg, respectively (single dose per cohort). Results of
the study showed that increases in reticulocyte count and
hemoglobin levels were achieved after the third cohort received 0.1
mg/kg of Peptide I. This dose was repeated in the fourth cohort, to
confirm the results observed in the third cohort. As such, the
study was terminated. Unblinded results are summarized below.
[0336] 2.2, Pharmacodynamic Results
[0337] Reticulocyte count (absolute number and percent) showed a
dose-dependent increase with increasing Peptide I dose.
Reticulocyte counts reached a maximum approximately 7 days postdose
in all dose cohorts. A comparison of the maximum reticulocyte
response, and reticulocyte vs. time curves (AUC.sub.0-14days and
AUC.sub.0-23days) showed significant differences among dose groups
(p<0.05).
[0338] The hemoglobin response and change from baseline (both mean
and maximum) over 28 days post-dose showed dose-dependent increases
with increasing dose of Peptide I whereas the control group
exhibited a slight decrease in hemoglobin over time secondary to
the study related blood draws without concomitant exogenous
stimulation of erythropoiesis [one-way Analysis of Variance (ANOVA)
p-value of 0.0001]. A one-way Analysis of Covariance (ANCOVA) using
mixed models was used to compare individual changes from baseline
over time among all four groups. The results of this analysis
showed a significant dose-response among all four dose groups
(p=0.0001), with significant differences between the 0.025 vs. 0.1
mg/kg groups (p=0.0027), as well as the 0.05 vs. 0.1 mg/kg groups
(p=0.0113). The PAD of Peptide I that was associated with at least
a 1.0 g/dL increase in hemoglobin was 0.1 mg/kg.
[0339] Additionally, subjects in Cohorts 3 and 4 (0.1 mg/kg Peptide
I or placebo) were followed through 42 days post-dose. At Day 42,
the average hemoglobin levels had returned to baseline in the
subjects treated with 0.1 mg/kg Peptide I, but were still below
baseline values in the placebo subjects. Thus, at Day 42, as
throughout the study, the difference in change from baseline
hemoglobin was .gtoreq.0.5 g/dL between subjects dosed with 0.1
mg/kg Peptide I versus those dosed with placebo.
[0340] Serum EPO levels transiently decreased with increasing
Peptide I dose. Changes in other pharmacodynamic parameters
(increased red cell count and hematocrit, transient decreases in
ferritin and reticulocyte hemoglobin content, transient increase in
soluble transferrin receptor protein, and transient decrease in
EPO) were consistent with stimulation of erythropoiesis.
[0341] 2.3. Pharmacokinetic Results
[0342] Following IV doses of 0.025, 0.05, and 0.1 mg/kg Peptide I
over a 5 minute period, drug concentrations generally peaked at
between 5 minutes and 1 hour after initiation of the infusion. In
the 0.025 mg/kg dose group, Peptide I concentration peaked between
5 and 15 minutes, while t.sub.max approached 1 hour for the 0.1
mg/kg dose. After C.sub.max was achieved, plasma concentrations
declined and were generally quantifiable (>25 ng/mL) up to 96
hours after the 0.025 mg/kg dose, up to Day 5 for the 0.05 mg/kg
dose, and up to Day 7 for the 0.1 mg/kg dose. Calculated half-lives
showed a small increase at the 0.1 mg/kg dose, with a range of:
16.7-21.9 hours (mean 19.2 hours) following the 0.025 mg/kg dose;
15.3-25.1 hours (mean 18.7 hours) following the 0.05 mg/kg dose;
and 17.7-33.1 hours (mean 23.5 hours) after the 0.1 mg/kg dose.
[0343] The drug's distribution and elimination appeared not to
follow standard one or two compartment kinetics. First order
kinetics were observed at lower drug concentrations only,
suggesting saturation of metabolic/elimination processes at plasma
concentrations in excess of approximately 400 ng/mL. Volume of
distribution showed little change with dose (means of 2165, 1903,
and 2010 mL for the 0.025, 0.05 and 0.1 mg/kg doses, respectively).
Plasma clearance showed a small decline with dose, with geometric
means of 78.0, 70.7 and, 59.2 mL/hr for the 0.025, 0.05, and 0.1
mg/kg doses, respectively. ANOVA of dose normalized C.sub.max, and
AUC.sub.(0-infinity) data showed that C.sub.max appeared to have a
linear relationship with dose. However, AUC.sub.(0-infinity) showed
evidence of non-linearity at the highest dose of 0.1 mg/kg,
possibly associated with the observed small decrease in drug
clearance at higher doses.
[0344] 2.4. Safety Results
[0345] Fifteen subjects (4 of 8 receiving placebo and 11 of 20
receiving Peptide I) experienced a total of 28 adverse events (AEs)
(6 in the placebo group and 22 in the Peptide I group). For
subjects dosed with Peptide I, headache (4/20, 20.0%) and abdominal
pain (2/20, 10.0%), nausea (2/20, 10.0%), and nasopharyngitis
(2/20, 10.0%) were the more frequently reported events. All AEs,
except one, were graded as mild (Grade 1). Most AEs observed among
the Peptide I recipients (15/22) were considered probably not
related to the study drug, Peptide I. There was no difference in
frequency, severity or pattern of AEs between the 4 groups. There
were no Serious Adverse Events (SAEs) or withdrawals from the study
due to an AE; however, study drug was discontinued for one subject
assigned to the 0.025 mg/kg dose of Peptide I due to a mild drug
reaction. This reaction which started within 2 minutes of the
infusion was a flush starting on the chest expanding to the face,
with a sensation of feeling hot, uncomfortable and a scratchy
throat. As a safety precaution, the infusion was discontinued for
this subject. Symptoms resolved spontaneously and rapidly. No
therapeutic intervention was required. There were no changes in
vital signs. Laboratory parameters were normal including additional
immunologic testing; therefore, the specific nature of this
reaction cannot be elucidated. Similar (or more severe) drug
reactions have been observed with many drugs, including other ESAs.
In addition, patients should be observed during IV administration
of Peptide I. An injection should be discontinued if a patient
develops similar symptoms.
[0346] One placebo recipient and one Peptide I recipient were given
concomitant medications for mild headache and mild abdominal
cramps, respectively. There were no clinically significant changes
in vital signs, electrocardiograms (ECGs) or laboratory values.
None of the subjects in this trial developed antibodies specific to
Peptide I.
[0347] 2.5. Summary
[0348] In summary, Peptide I appeared safe and well tolerated after
single IV doses of 0.025, 0.05, or 0.1 mg/kg, with a safety profile
similar to placebo. The pharmacokinetic results showed a halflife
ranging from approximately 15 to 33 hours, with a mean of 23.5
hours at the 0.1 mg/kg dose. The median was comparable for all
doses, occurring 15 minutes after the start of the infusion.
C.sub.max appeared to have a linear relationship with dose;
AUC.sub.(0-infinity) appeared to be nonlinear at the 0.1 mg/kg
dose. First order kinetics were observed at lower drug
concentrations only, suggesting saturation of metabolic/elimination
processes at plasma concentrations >400 ng/mL. Peptide I showed
pharmacological activity for reticulocytes at all doses evaluated;
generally the responses were dose-dependent with greater and longer
responses with increasing dose. The 0.1 mg/kg dose group was
defined as the PAD in NHV as it was associated with a clinically
and statistically significant increase in hemoglobin from baseline,
with an average maximum increase from baseline of 1.36.+-.0.39 g/dL
among the 10 Peptide I recipients. Changes in other pharmacodynamic
parameters (increased red cell count and hematocrit, transient
decreases in ferritin and reticulocyte hemoglobin content,
transient increase in soluble transferrin receptor protein, and
transient decrease in EPO) were consistent with stimulation of
erythropoiesis.
[0349] Incorporated herein by reference in its entirety is the
poster and abstract #0470 presented at the European Hematology
Association 10th Annual Congress in Stockholm on Jun. 4, 2005.
Example 14
Increase in Hemoglobin Levels of Pre-dialysis, Dialysis, and
Oncology Patients
1. Pre-Dialysis Patients
[0350] A randomized, double blind, placebo controlled, sequential
dose escalation study of the safety, pharmacodynamics, and
pharmacokinetics of single intravenous doses of Peptide I injection
in patients with chronic kidney disease (CKD) who are not on
dialysis and who have not had prior erythropoiesis stimulating
agent (ESA) treatment will be conducted. This study will evaluate
the safety profile of single intravenous (IV) dose levels of
Peptide I in CKD patients not on dialysis (pre dialysis patients).
This study will also: evaluate the dose response relationships of a
single dose of Peptide I on pharmacodynamic parameters including
hemoglobin, reticulocytes, and iron stores; evaluate the
pharmacokinetic profiles of single dose levels of Peptide I
intravenously in pre dialysis patients; determine the
pharmacologically active dose (PAD) intravenously in pre dialysis
patients, e.g., the dose that results in >70% of patients
achieving a .gtoreq.1 g/dL hemoglobin increase from baseline
[0351] The endpoints of the study will include: adverse events
(AEs); serious adverse events (SAES); pharmacokinetic parameters
including C.sub.max, AUC.sub.0-t AUC.sub.0-infinity,
t.sub.1/2.beta., Vd, and CL; pharmacologic parameters including
reticulocytes, hemoglobin, reticulocyte hemoglobin content, and
serum measures of iron stores (e.g., serum ferritin, transferrin
saturation, and transferrin receptor protein); average hemoglobin
change from baseline; maximum hemoglobin change from baseline;
proportion of treated patients who achieve a >1 g/dL hemoglobin
increase from baseline; and frequency of red blood cell
transfusions.
[0352] The patients selected for the study will be those
pre-dialysis patients aged 18-75 years with hemoglobin .gtoreq.9
g/dL and .ltoreq.11 g/dL secondary to chronic kidney disease who
have not had previous treatment with ESAs and who meet eligibility
criteria will be enrolled. In each cohort of 9 patients, patients
will be randomly assigned to receive a single dose of Peptide I
(n=7) or placebo (n=2). To ensure availability of data for a
minimum of 22 days in 5 Peptide I treated patients per cohort, the
third and subsequent patients in a cohort who terminate study prior
to day 22 will be replaced. Each replacement patient will be
assigned to the same treatment group as the withdrawn patient.
Patients who terminate from study after day 22 will not be
replaced.
[0353] Up to 6 dose level cohorts (up to 4 planned dose levels and
LIP to 2 additional lower, intermediate, and/or repeat
[confirmatory] dose levels) are planned to be sequentially enrolled
as determined by the Independent Safety Monitor, Investigator, and
Sponsor. A maximum of 54 evaluable patients may be enrolled in this
trial.
[0354] Each patient in the study is expected to participate for at
least 28 days following dosing.
[0355] Each patient will receive a single dose of Peptide I or
placebo. Planned sequential Peptide I dose levels are:
TABLE-US-00011 Peptide I Dose (mg/kg) 0.05 0.1 0.2 0.3 Additional
Confirmatory, Intermediate, or Lower Dose Additional Confirmatory,
Intermediate, or Lower Dose
[0356] This is a randomized, double blind, placebo controlled,
sequential dose escalation study with up to G dose cohorts of 9 pre
dialysis patients per cohort. In each cohort, patients will be
followed through day 29, or until stabilization of adverse events,
whichever occurs later. Hemoglobin levels will be followed in all
patients until elevated levels return to within 0.5 g/dL of
baseline levels.
[0357] Baseline hemoglobin concentration is defined as the mean of
the three most recent hemoglobin values prior to study drug dosing.
During the study, changes in hemoglobin for a cohort initiation or
stopping decision require confirmation by the next consecutive
hemoglobin value.
[0358] If a patient's hemoglobin level reaches 14 g/dL, the patient
should be phlebotomized if clinically indicated. If a patient's
hemoglobin level reaches 16 g/dL (confirmed), the patient should be
phlebotomized.
[0359] After dosing of the first cohort, subsequent enrollment of
the next cohort at the next dose level will be based on protocol
specified dose escalation criteria. Dose escalation to the next
dose level cohort will be halted based on protocol specified
stopping criteria. An unblinded, Independent Safety Monitor will
review the unblinded clinical and laboratory data on an ongoing
basis to determine when the dose escalation or stopping criteria
have been met.
[0360] The Investigator clinic personnel and Sponsor clinical
personnel will be blinded to individual patient treatment
assignment and patient identified PK, hematologic, and iron
parameter results including hematocrit, hemoglobin, MCHC (mean
corpuscular hemoglobin concentration), MCH (mean corpuscular
hemoglobin), MCV (mean corpuscular volume), reticulocyte count, and
reticulocyte hemoglobin content, or iron store results including
ferritin, transferrin saturation, and transferrin receptor protein;
however, these personnel may review de identified results for these
parameters during the course of the study. These personnel will not
have access to patient identified results for these parameters, as
the results, if identified by patient, could potentially unblind to
treatment assignment.
[0361] Beginning when all evaluable patients (no less than 7) in a
cohort reach day 22, dose escalation to the next cohort is allowed
if no safety concerns are identified and: no patient in the cohort
achieves a hemoglobin increase .gtoreq.1.0 g/dL; or all patients
who receive Peptide I and achieve a hemoglobin increase >1.0
g/dL from baseline demonstrate either stability (within 0.5 g/dL of
peak hemoglobin value) or reversibility (decrease from peak
hemoglobin value) over a minimum of two time points.
[0362] Enrollment of new patients into the current cohort and
escalation to a new dose will be stopped if any of the following
criteria are met: Two or more patients in a cohort have a Grade 3
or 4 Peptide I related adverse event; two or more patients who
receive Peptide I in a cohort have a .gtoreq.2.0 g/dL increase in
hemoglobin (increase starting above baseline level) over any 4 week
period; or two or more patients who receive Peptide I in a cohort
exceed the upper limit of the target range of hemoglobin (>13
g/dL).
[0363] If no safety concerns are identified, additional cohorts of
9 patients may be initiated after the all evaluable patients (no
less than 7) in a cohort have reached day 22 to study confirmatory
(repeat), lower, or intermediate doses.
[0364] It is expected that for the pre-dialysis patients, a PAD of
0.025 to 0.2 mg/kg, possibly 0.05-0.1 mg/kg, possibly 0.067 to
0.075 mg/kg, will be determined.
2. Dialysis Patients
[0365] An open-label, multi-center, sequential, dose finding study
of the safety, pharmacodynamics, and pharmacokinetics of Peptide I
injection administered intravenously for the maintenance treatment
of anemia in chronic hemodialysis patients will be performed to
determine the range of monthly intravenously administered Peptide I
doses that maintains hemoglobin within 1.0 g/dL above or below
baseline in hemodialysis patients whose hemoglobin values were
stable on Epoetin alfa. This study will also evaluate the safety
profile of up to 3 doses of Peptide I administered intravenously in
hemodialysis patients; evaluate the pharmacokinetic profile of up
to 3 doses Peptide I administered intravenously in hemodialysis
patients (in a subset of study patients).
[0366] The endpoints of the study include: average weekly
hemoglobin change from baseline; number (%) of patients with
hemoglobin within 1.0 g/dL above or below baseline through Week 13;
number (%) of patients who maintain hemoglobin within 9.5-13.0 g/dL
through Week 13; number (%) of patients with no dose adjustments
during the study and number (%) of patients with dose increase or
decrease during the study; frequency of red blood cell
transfusions; additional pharmacologic parameters including
reticulocyte count (absolute and AUC), reticulocyte hemoglobin
content, and serum measures of iron stores (e.g., serum ferritin,
transferrin saturation, and soluble transferrin receptor protein);
adverse events; serious adverse events; and pharmacokinetic
parameters including C.sub.max, AUC.sub.0-t AUC.sub.O-infinity,
t.sub.1/2.beta., Vd, Vss and CL (in a subset of study patients).
Results of the pharmacokinetic and pharmacodynamic analyses will be
used to determine the preliminary conversion factor that best
predicts the monthly dose of Peptide I based on the weekly dose of
Epoetin alfa.
[0367] The patients selected for the study will be those
hemodialysis patients aged 18 years or older with stable hemoglobin
maintained on a stable dose of commercially available Epoetin alfa
and who meet eligibility criteria will be enrolled. Up to 4
sequential dose level cohorts of 15 patients per cohort are planned
to be enrolled at 3 to 10 clinical centers.
[0368] To ensure availability of data in a minimum of 10 patients
per cohort who have received 3 Peptide I doses, the 6th and
subsequent patients in a cohort who terminate study prior to
receiving the 3rd dose will be replaced, up to a maximum of 4
replacements. Patients who terminate from study after receiving 3
doses will not be replaced.
[0369] Two dose level cohorts are initially planned to be
sequentially enrolled. Depending on the observed safety profile and
pharmacologic response, up to 2 additional cohorts of 15 patients
each may be added to study lower, intermediate, and/or repeat
(confirmatory) dose levels, and/or frequency of administration.
[0370] A minimum of 30 and a maximum of 60 evaluable patients may
be enrolled in this trial.
[0371] Each patient in the study is expected to participate for
approximately 15 weeks following a 4 week screening period.
[0372] An amendment to lengthen the dosing duration for an
additional 12 weeks is planned pending availability of supportive
data.
[0373] Doses of Peptide I will be administered every 4 weeks for a
total of three doses as rapid intravenous bolus injections over 30
seconds during the last 15 minutes of dialysis. Each patient in a
cohort will receive open-label doses of Peptide I starting at a
pre-specified Epoetin alfa-to-Peptide I conversion level. The dose
will be escalated or de-escalated for the next cohort based on the
dose response relationship observed in the previous cohort. The
planned starting Peptide I dose level conversions are:
TABLE-US-00012 100 U/kg/week Epoetin alfa to Peptide I Cohort Dose
(mg/kg/Q4W) Conversion Factor Starting Cohort 0.033 Escalation or
De- 0.041-0.050 for escalation escalation Cohort 0.017-0.025 for
de-escalation Intermediate or To be determined Confirmatory
Cohort(s) Intermediate or To be determined Confirmatory Cohorts
[0374] The Peptide I dose may be adjusted in individual patients as
follows. Starting with the 3.sup.rd dose of the Peptide I study
drug, the dose will be increased by 25% if a patient's confirmed
hemoglobin decreases by >1.0 g/dL from baseline or a patient's
confirmed hemoglobin decreases by .gtoreq.0.5 g/dL from baseline to
a level below 11 g/dL. Starting with the 3.sup.rd dose of study
drug, the dose will be reduced by 25% if a patient's confirmed
hemoglobin increases by >1.5 g/dL from baseline or a patient's
confirmed hemoglobin increases by .gtoreq.0.5 g/dL from baseline to
a level above 12 g/dL. The next dose will be reduced by 25% if at
any time during the study a patient's confirmed hemoglobin
increases (increase starting above baseline) by >1.0 g/dL within
any 2 week period. If at any time during the study, a patient's
confirmed hemoglobin exceeds 12.5 g/dL, the next dose will be
delayed until the hemoglobin decreases to 12.0 g/dL and the dose
will be reduced by 25%.
[0375] Baseline hemoglobin concentration is defined as the mean of
the 3 most recent mid-week pre-dialysis hemoglobin values collected
in the 3 weeks prior to administration of study drug. During the
study, changes in hemoglobin level for individual dose adjustment
or cohort initiation or stopping criteria decisions require
confirmation with a repeat hemoglobin value at any time within 7
days.
[0376] This is an open-label, sequential dose finding trial with
2-4 treatment cohorts of 15 hemodialysis patients per cohort. Each
patient will receive an intravenous dose of Peptide 1 every 4 weeks
for a total of 3 doses. After receiving the first dose of Peptide
I, patients will be seen at least weekly throughout the study.
During the study, iron status will be maintained per Kidney Disease
Outcomes Quality Initiative (K/DOQI) treatment guidelines.
[0377] Patients will be followed for a minimum of 42 days after the
last administration of Peptide I, or until stabilization of adverse
events, whichever occurs later. After ceasing treatment, hemoglobin
levels will be followed in all patients until return to target
range.
[0378] If a patient's hemoglobin level reaches 14 g/dL (confirmed),
the patient may be phlebotomized per the Investigator's judgment.
If a patient's hemoglobin level reaches 16 g/dL (confirmed), the
patient will be phlebotomized. The method of phlebotomy will be
done per the site's standard clinical practice. The volume of blood
phlebotomized will be documented. Phlebotomized patients will
discontinue receipt of Peptide I and postphlebotomy pharmacodynamic
data will be excluded from analysis.
[0379] After dosing of the first cohort, subsequent enrollment of
the next cohort is based on protocol-specified dose escalation and
de-escalation criteria. Once an appropriate dose level conversion
factor is identified, the conversion factor dose level may be
repeated in a confirmatory cohort. The Independent Safety Monitor
and Sponsor will review the clinical and laboratory data on an
ongoing basis to determine when the dose escalation, de-escalation,
additional cohort, or stopping criteria have been met.
[0380] Beginning at the Week 7 data review of 6 or more patients
enrolled in a cohort, enrollment into the current cohort may be
stopped and dose escalation to the next cohort is allowed if no
safety concerns are identified by the Independent Safety Monitor
and a combined 6 or more patients have a confirmed hemoglobin
decrease from baseline of more than 1.0 g/dL at Week 7 or
later.
[0381] Beginning at the Week 7 data review of 6 or more patients
enrolled in a cohort, enrollment into the current cohort may be
stopped and dose de-escalation to the next cohort is allowed if no
safety concerns are identified by the Independent Safety Monitor
and a combined 6 or more patients have a confirmed hemoglobin
increase >1.0 g/dL from baseline to a hemoglobin value >13.0
g/dL occurring at Week 7 or later or a confirmed hemoglobin
increase >1.0 g/dL (increase starting above baseline) within any
two week period after study entry.
[0382] Beginning at the Week 7 data review of 10 or more patients
enrolled in a cohort, if no safety concerns are identified by the
Independent Safety Monitor, an additional cohort may be initiated
to study lower or intermediate (between the current and previously
studied) conversion factors, and/or frequency of administration. A
maximum of two additional cohorts may be enrolled.
[0383] After the Week 7 data review of 10 or more patients enrolled
in a cohort, a confirmatory cohort utilizing the same conversion
factor may be initiated to repeat the same conversion factor and/or
frequency of administration if no safety concerns are identified by
the Independent Safety Monitor and neither dose escalation nor dose
de-escalation rules have been met.
[0384] Enrollment of new patients into the current cohort and
escalation to a new dose will be stopped if the following criterion
is met: 3 patients in a cohort have a Grade 3 or Grade 4 Peptide
1-related adverse event.
[0385] It is expected that for the dialysis patients, a PAD of
0.025 to 0.2 mg/kg, possibly 0.05-0.1 mg/kg, possibly 0.067 to
0.075 mg/kg, will be determined.
3. Oncology Patients
[0386] An open-label, multi-center dose-escalation study of the
safety, pharmacodynamics, and pharmacokinetics of subcutaneously
administered Peptide I in cancer patients with chemotherapy induced
anemia (CIA) will be conducted to determine the Peptide I dose to
be administered every three weeks by subcutaneous (SC) injection
associated with a hemoglobin response in patients with chemotherapy
induced anemia. Other objectives include to: evaluate the safety
profile of up to 4 doses of Peptide I administered subcutaneously
every three weeks in cancer patients receiving concomitant
myelosuppressive chemotherapy; determine the change from baseline
in Hgb in patients with CIA at different dose levels of Peptide I;
determine the proportion of patients who have a Hgb response to
Peptide I (as defined in Endpoints); determine the dose of Peptide
I administered subcutaneously that increases and maintains the
hemoglobin in the target range of 11-13 g/dL in CIA patients;
evaluate the pharmacokinetic profile of up to 4 doses of Peptide I
administered subcutaneously in CIA patients (in a subset of study
patients); and explore the effect of dose frequency and parenteral
iron replacement at an active dose of Peptide I.
[0387] The endpoints of the study will include: proportion of
patients per treatment group who have > a 2 g/dL increase in
hemoglobin OR who have an increase of Hgb of >1 g/dL to at least
12 g/dL at 4 weeks, 9 weeks and 12 weeks in the absence of RBC
transfusion in the previous 28 days; proportion of patients who
have a Hgb increase of >1 g/dL from baseline at 4 weeks, 9 weeks
and 12 weeks in the absence of RBC transfusion in the previous 28
days; proportion of patients who have a Hgb in the target range of
11-13 g/dL at weeks 4, 9 and 12; proportion of Hgb values in the
target range of 11-13 g/dL after week 4; average hemoglobin change
from baseline; frequency of red blood cell transfusions; additional
pharmacologic parameters including reticulocyte counts (absolute
and AUC), reticulocyte hemoglobin content; changes in measures of
iron stores, e.g. transferrin saturation and serum ferritin;
adverse events (AEs); serious adverse events (SAEs); and in a
subset of study patients pharmacokinetic parameters including
C.sub.max, AUC.sub.0-t, AUC.sub.0-.infin., t.sub.1/2.beta.
(elimination half-life), Vd (apparent volume of distribution), Vss
(steady-state volume), and CL (clearance).
[0388] The patients selected for the study will be those with solid
tumors or lymphoma, aged 18-80 years with hemoglobin .gtoreq.9 g/dL
and .ltoreq.11 g/dL secondary to chemotherapy who have not had
previous treatment with ESAs within the past 90 days and who meet
eligibility criteria and will be assigned to Peptide I at the
starting dose or subsequent sequential dose level approved by the
Medical Monitor (MM). To ensure availability of 12 week data in of
a minimum 10 treated patients per treatment group, the 6th and
subsequent patients in a group who terminate the study prior to 8
weeks, will be replaced, up to a maximum of 5 replacements. Each
replacement patient will be assigned to the same treatment group as
the withdrawn patient. Up to 4 open-label treatment groups of 15
patients may be subsequently added to study lower, intermediate,
and/or repeat dose levels and/or frequency of administration of
Peptide I and/or parenteral iron replacement to maintain
transferrin saturation levels at 25-50%. A minimum of 30 and a
maximum of 90 patients (not including replacements) may be enrolled
in this trial if all possible dosing regimens are explored.
[0389] Each patient in the study is expected to participate for up
to 12 weeks following up to a 4 week screening period.
[0390] Sequential cohorts of fifteen patients will receive
escalating doses of Peptide I. Open-label doses will be
administered by SC injection every 3 weeks for a total of four
doses (Study weeks 1, 4, 7, 10). Typically, Peptide I would be
administered on Day 1 of a chemotherapy cycle. The Peptide I
starting doses and frequency of dosing are based on Phase 1 data in
Healthy Volunteers as well as predicted responses modeled from
PK/PD data of the erythropoietic response to Peptide I and other
Erythropoiesis Stimulating Agents (ESAs). Planned Peptide dose
levels and the number of patients in each group are show in Table
7:
TABLE-US-00013 TABLE 7 Peptide I Dose Levels and Patient Numbers N
Treatment Dose (mg/kg) Q3W 15 Peptide I 0.1 mg/kg 15 Peptide I 0.2
mg/kg 15 Peptide I 0.4 mg/kg 15 Peptide I 0.6 mg/kg
Patients will be followed for 28 days post last injection, or until
stabilization of adverse events, or hemoglobin values are between
11-13 g/dL which ever comes last. No dose increases will be
allowed. After week 4, if RBC transfusions are required to maintain
the Hgb in the target range, the patient will be removed from study
drug, return to standard of care, and be followed for an additional
28 days for safety. If at any time during the study, a patient's
hemoglobin increases by >1.0 g/dL within any 2 week period, the
next dose will be delayed until the hemoglobin stabilizes (an
increase of <0.5 g/dL in a week) and the dose will be reduced by
50%. Baseline hemoglobin concentration is defined as the mean of
the 2 most recent weekly hemoglobin values collected in the week
prior to administration of study drug (e.g. screening and baseline
values). During the study, changes in hemoglobin level for cohort
initiation or stopping criteria decisions require confirmation by
the next consecutive hemoglobin value within 7 days.
[0391] This is an open-label, multi-center trial with up to 6
treatment groups of 15 patients with CIA per group. In the initial
treatment groups, open-label Peptide I will be administered by
subcutaneous injection every 3 weeks for a total of up to 4 doses.
After the first dose, patients will be seen at least weekly
throughout the study. Patients will be followed for a minimum of 28
days after the last administration of study drug, or until
stabilization of adverse events, or hemoglobin values are between
11-13 g/dL whichever occurs last. If a patient's hemoglobin level
reaches 14 g/dL (confirmed), the patient may be phlebotomized if
clinically indicated and the volume of phlebotomy documented.
Phlebotomized patients will discontinue receipt of study drug and
post-phlebotomy pharmacodynamic data will be excluded from
analysis. The Medical Monitor (MM), and Sponsor will review the
safety and pharmacodynamic data on an ongoing basis to determine if
and when the stopping criteria have been met.
[0392] After 6 or more patients enrolled in a cohort have completed
at least 6 weeks of follow up (i.e. at least three weeks following
the second dose), enrollment into the current cohort may be stopped
and dose escalation to the next cohort is allowed if there are: no
safety concerns are identified by the MM; and, 6 or more patients
are transfused after week 4 or have a confirmed hemoglobin increase
of <1 g/dL at week 6.
[0393] After 6 or more patients enrolled in a cohort and have
completed at least 6 weeks of follow up (at least three weeks
following the second dose), enrollment into the current cohort will
be stopped and dose de-escalation to a lower level in the next
cohort may be allowed if there is: an occurrence of at least 3
Grade 3 or 4 AEs possibly related to Peptide I or if the MM
identifies any specific concern to Peptide I; or a total of 6 or
more patients have a confirmed hemoglobin level >13.0 g/dL (not
related to transfusion), or a confirmed hemoglobin increase >1.0
g/dL within any two week period (not related to transfusion)
[0394] If no safety concerns are identified by the MM and Sponsor,
up to two additional open-label treatment groups of 15 patients may
be initiated to study lower, intermediate (between the current and
previously studied), or repeat dose levels. In addition, once the
active dose administered every three weeks is determined, up to 2
additional cohorts may be enrolled to determine the relative effect
of the same total dose administered in fractions at a different
frequency (e.g., 4/3 of the every 3 week dose administered every 4
weeks). The effect of administration of parenteral iron to achieve
a transferrin saturation of 25-50% may also be explored in a
separate cohort.
[0395] It is expected that for the oncology patients, a PAD of
0.075-0.5 mg/kg, possibly 0.2-0.4 mg/kg, possibly 0.25 mg/kg, will
be determined.
Example 15
Long-Term Safety, Tolerability, and Pharmacodynamics of Peptide I
in a Phase II, Multi-Dose Study in Patients with Chronic Kidney
Disease
1. Study Methods
[0396] A multi-center, open-label, sequential dose-finding phase II
clinical trial involving patients with chronic kidney disease was
performed in order to evaluate the safety and pharmacodynamics of
multiple doses of once-monthly (Q4W) subcutaneous (SC) delivery of
Peptide I (SEQ ID NO:2). A total of 60
erythropoiesis-stimulating-agent-naive, pre-dialysis, chronic
kidney disease (CKD) patients [hemoglobin (Hgb) levels 9.0-10.9
g/dL, ferritin >100 ug/L and transferrin saturation
(TSAT)>20%] were enrolled into three dose cohorts. Patients
received up to six once-monthly SC drug doses. The starting doses
were 0.025 mg/kg (n=15), 0.050 mg/kg (n=30) and 0.075 mg/kg (n=15).
Dose titration was allowed after the first dose, based on patient
Hgb levels.
2. Mean Baseline Characteristics and Individual Patient Dose
Adjustments
[0397] Baseline characteristics were balance across dose groups as
shown in the Table 8:
TABLE-US-00014 TABLE 8 Dose Cohort (range) 0.025 mg/kg 0.050 mg/kg
0.075 mg/kg All Doses (n = 15) (n = 30) (n = 15) (n = 60) Gender
(M:F) 9:6 (60% M) 22:8 (73% M) 11:4 (73% M) 42:18 (73% M) Age (yr)
64 (13) 65 (12) 65 (14) 65 (13) Weight (kg) 85 (26) 79 (14) 81 (14)
81 (18) Hgb (g/dL) 10.2 (0.5) 10.4 (0.5) 10.2 (0.5) 10.3 (0.5) TSAT
(%) 26 (8) 27 (7) 30 (12) 28 (9) Ferritin (mcg/L) 245 (162) 293
(226) 271 (222) 276 (208)
[0398] Dose titrations were allowed after the patient's initial
dose. Dose adjustments were .+-.25% of the previous dose, depending
on the patient's measured hemoglobin (Hgb) level. Patients (shown
by percent) that were dose-titrated by week 12, are shown in Table
9.
TABLE-US-00015 TABLE 9 Patients Dose-titrated by Week 12 Dose
Cohort Dose 0.025 mg/kg 0.050 mg/kg 0.075 mg/kg Adjustment (n = 15)
(n = 30) (n = 15) Dose Increase 6 (40%) 3 (10%) 0 (0%) Dose
Decrease 1 (7%) 10 (33%) 5 (33%)
[0399] The study pharmacodynamic results are shown in FIGS. 1-4.
The mean reticulocyte change from baseline for 0-12 weeks is shown
in FIG. 1. The mean hemoglobin (Hgb) change from baseline for the
study weeks 0-12 is shown in FIG. 2. The correction of anemia
(Hgb>11 g/dL) by dose and treatment duration is shown in FIG. 3
depicts. The mean hemoglobin (Hgb) change from baseline for the
study weeks 12-22 is shown in FIG. 4. The hemoglobin (Hgb) change
from baseline for individual patients in the 0.05 mg/kg cohort for
the study weeks 0 to 12 is shown in FIG. 5.
3. Safety Results
[0400] Adverse events and serious adverse events were monitored
during the study. Eleven (18%) patients reported 36 adverse events.
All events were assessed as not related to study drug. No adverse
event resulted in study withdrawal, no injection site reaction was
reported. All serious adverse events were assessed as not related
to study drug.
4. Conclusions
[0401] Pre-dialysis chronic kidney disease (CKD) patients can be
effectively treated with Peptide I by monthly dosing via a
subcutaneous injection. Multiple monthly subcutaneous Peptide I
injections are well-tolerated. Monthly drug dosing achieves
correction of anemia in patients by week eight. The drug results in
a sustained increase in hemoglobin (Hgb) up to week 22, when dosed
monthly in patients with chronic kidney disease.
Example 16
Safety, Tolerability, and Pharmacodynamics of Peptide I in a Phase
II Multi-Dose Study in Patients Suffering from Anemia Associated
with Solid Tumor Malignancy or Lymphoma
1. Study Methods
[0402] A multi-center, open label, sequential dose-finding phase II
clinical trial involving patients with cancer was performed in
order to evaluate the safety and pharmacodynamics of multiple doses
administered every 3 weeks (Q3W) subcutaneous (SC) delivery of
Peptide I (SEQ ID NO: 2). A total of 60 patients with solid tumor
malignancy or lymphoma [patients had greater than 9 weeks of
chemotherapy and baseline hemoglobin levels greater than 8 g/dL but
less than 11 g/dL and adequate iron, folate and B12 stores] were
enrolled into four dose cohorts. Patients received up to two SC
drug doses which were given once every three weeks. The starting
doses were 0.05 mg/kg (n=15), 0.10 mg/kg (n=15), 0.15 mg/kg (n=15),
and 0.20 kg/mg (n=15).
2. Mean Baseline Characteristics and Pharmacodynamic Results
[0403] Mean baseline hemoglobin values and the percent of patients
completing the study across the four dose cohorts are shown in the
following table. Table 10 below also shows the percent of patients
at Week 7 of the study showing a mean increase from baseline
hemoglobin of .gtoreq.1 g/dL in the pharmacodynamic dataset
comprising 42 patients across the four dose cohorts.
TABLE-US-00016 TABLE 10 Percent of Patients at Week 7 with Mean
Increase from Baseline Hemoglobin 0.05 mg/kg 0.10 mg/kg 0.15 mg/kg
0.20 kg/mg (n = 15) (n = 15) (n = 15) (n = 15) Mean 10.1( .+-.
1.07) 10.0( .+-. 0.89) 9.8( .+-. 0.67) 10.1( .+-. 0.69) Baseline
Hemoglobin Level (g/dL) Approximate 79 50 60 92 Percent of Patients
Completing the Study (%) Percent (%) 20 70 55 55 of Patients
Showing a Mean Increase From Baseline Hemoglobin of .gtoreq. 1
g/dL
3. Safety Results
[0404] Adverse events and serious adverse events were monitored
during the study. Three patients withdrew from the study due to
adverse events that were assessed as not related to the study drug.
Six patients withdrew from the study due to serious adverse events.
One serious adverse event, thrombophlebitis, was probably related
to the study drug; however the other five reports of serious
adverse events were not attributed to the study drug. Three patient
deaths occurred during the course of the drug study (two deaths due
to disease progression and one death due to renal insufficiency).
None of the deaths was attributed to the study drug.
4. Conclusions
[0405] Patients suffering from anemia associated with solid tumor
malignancy or lymphoma who are also undergoing chemotherapy can be
effectively treated with Peptide I by subcutaneous injection every
three weeks. Subcutaneous injections of Peptide I every three weeks
was well tolerated. By week 7 of the study, Peptide I resulted in a
mean increase from baseline hemoglobin of .gtoreq.1 g/dL in
patients of the study.
Example 17
Safety, Tolerability, and Pharmacodynamics of Peptide I in a Phase
II Multi-Dose Study in Patients Suffering from Anemia Associated
with Solid Tumor Malignancy or Lymphoma
1. Study Methods
[0406] A multi-center, open label, sequential dose-finding phase II
clinical trial involving patients with cancer was performed in
order to evaluate the safety and pharmacodynamics of multiple doses
administered Q3W SC delivery of Peptide I (SEQ ID NO: 2). A total
of 60 patients with solid tumor malignancy or lymphoma [patients
had greater than 9 weeks of chemotherapy and baseline hemoglobin
levels greater than 8 g/dL but less than 11 g/dL and adequate iron,
folate and B12 stores and ECOG performance status of 0-2] were
enrolled into four dose cohorts. Patients received at least two SC
drug doses which were given once every three weeks. The starting
doses were 0.05 mg/kg (n=15), 0.10 mg/kg (n=15), 0.15 mg/kg (n=15),
and 0.20 kg/mg (n=15).
2. Mean Baseline Characteristics and Pharmacodynamic Results
[0407] The pharmacodynamic data set (n=49) included patients who
received at least two (2) doses of Peptide I and had at least two
(2) hemoglobin values at or after six (6) weeks following the first
dose. Hemoglobin responses were determined in patients without red
blood cell transfusions in the previous twenty-eight (28) days.
Demographic and baseline characteristics for patients completing
the study across the four dose cohorts are shown in the Table
11.
TABLE-US-00017 TABLE 11 Patient Demographic and baseline
characteristics 0.05 mg/kg 0.1 mg/kg 0.15 mg/kg 0.2 mg/kg Q3W Q3W
Q3W Q3W All Patients Parameter (N = 15) (N = 15) (N = 15) (N = 15)
(N = 60) Gender (M:F) 8:7 7:8 6:9 6:9 27:33 Age (yr) - mean .+-. SD
58 .+-. 11 64 .+-. 7 60 .+-. 9 59 .+-. 10 60 .+-. 9 Weight (kg) -
mean .+-. SD 71 .+-. 20 68 .+-. 10 72 .+-. 15 76 .+-. 18 72 .+-. 16
Hb (g/dL) - mean .+-. SD 10.2 .+-. 0.9 10.0 .+-. 0.9 9.6 .+-. 0.9
10.1 .+-. 0.7 10.0 .+-. 0.9 TSAT (%) - mean .+-. SD 25 .+-. 20 21
.+-. 7 23 .+-. 11 23 .+-. 13 23 .+-. 13 Ferritin (.mu.g/L) - median
(range) 266 (44-1752) 298 (61-744) 598 (22-3611) 292 (20-2027) 324
(20-3611) Solid tumor:lymphoma 15:0 13:2 11:4 14:1 53:7 Use of
Platinum compounds - n (%) 10 (67%) 5 (33%) 5 (33%) 8 (53%) 28
(47%) Use of Taxanes - n (%) 1 (7%) 1 (7%) 3 (20%) 5 (33%) 10
(17%)
[0408] FIG. 6 depicts an increase in hemoglobin from baseline of
.gtoreq.1 g/dL at 6 weeks following the first dose of 0.5 mg/kg,
0.1 mg/kg, 0.15 mg/kg, and 0.2 mg/kg of Peptide I (SEQ ID NO: 2)
was observed in 17%, 73%, 55%, and 47% of patients, respectively.
FIG. 7 depicts the percent of patients at Week 6 after the first
dose of the study showing a hemoglobin increase of .gtoreq.2 g/dL
or the combination of a .gtoreq.1 g/dL increase and a hemoglobin
value of at least 11 g/dL occurred in 8%, 73%, 55%, and 40% of
patients in the 0.5 mg/kg, 0.1 mg/kg, 0.15 mg/kg, and 0.2 mg/kg
dose cohorts, respectively. As shown in Table 12, dose reductions
and delays to occurred more frequently in the 0.2 mg/kg cohort:
TABLE-US-00018 TABLE 12 0.05 mg/kg 0.1 mg/kg 0.15 mg/kg 0.2 mg/kg
Q3W Q3W Q3W Q3W Parameter (N = 12) (N = 11) (N = 11) (N = 15)
Patients with 2 (17%) 0 1 (9%) 3 (20%) transfusions on or before
Dose 3 Patients with at least 1 (8%) 2 (18%) 0 5 (33%) 1 dose
reduction Patients with at least 1 dose delay 1 (8%) 2 (18%) 0 4
(27%)
[0409] FIG. 8 depicts that the patients in the 0.1 mg/kg, 0.15
mg/kg, and 0.2 mg/kg dose cohorts had increases in hemoglobin from
baseline.
3. Safety Results
[0410] Adverse events and serious adverse events were monitored
during the study. Forty-five (45) of the sixty (60) patients
reported an adverse event. The most frequently reported adverse
events were; nausea (22%), pyrexia (12%), vomiting (12%), anemia
(10%), dyspnea (10%), constipation (8%), febrile neutropenia (8%).
Eight (8) patients (representing 13%) had one or more of the
adverse events considered related to the study drug. In two (2)
patients, the following five events occurred: constipation, nausea,
vomiting, abnormal hematology test, and increased reticulocyte
count. One (1) patient suffered eleven (11) other related
events.
[0411] Thirty-seven (37) serious adverse were reported in eighteen
(18) patients. One serious adverse event, thrombophlebitis, was
probably related to the study drug; however the other reports of
serious adverse events were not attributed to the study drug. The
most frequently reported serious adverse events included: febrile
neutropenia (8%), neutropenia (5%), neutropenic sepsis (5%),
pancytopenia (5%), anemia (3%), and infection (3%). Three patient
deaths due to serious adverse events occurred during the course of
the drug study (one death due to pulmonary edema, one death due to
disease progression and one death due to renal insufficiency). A
fourth death occurred >one (1) month after study completion due
to serious adverse events of pleural effusion and decreased
performance status. None of the deaths was attributed to the study
drug.
4. Conclusions
[0412] Patients suffering from anemia associated with solid tumor
malignancy or lymphoma who are also undergoing chemotherapy can be
effectively treated with Peptide I by subcutaneous injection every
three weeks. Subcutaneous injections of Peptide I every three weeks
was well tolerated. By week 6 of the study, Peptide I at doses of
0.1 mg/kg and 0.15 mg/kg administered by subcutaneous injection
every three weeks in oncology patients also undergoing
myelosuppressive chemotherapy resulted in an increase of hemoglobin
of .gtoreq.1 g/dL in 50% of patients of the study. The increase in
hemoglobin in the 0.1 mg/kg and 0.15 mg/kg dose cohorts appeared to
be sustained through nine (9) and twelve (12) weeks. The 0.05 mg/kg
dose did not produce a meaningful increase in hemoglobin; however,
hemoglobin values were maintained close to baseline. While
forty-seven percent (47%) of the patients in the 0.2 mg/kg dose
cohort had a hemoglobin increase of .gtoreq.1 g/dL by week 6, this
was not sustained. The trend toward a lower response rate and a
lower average hemoglobin over time in 0.2 mg/kg dose cohort
compared to the 0.1 mg/kg or 0.15 mg/kg dose cohorts is due to
several factors, including: a greater number of dose delays and
reductions secondary to initial substantial hemoglobin
increases.
Hgb and Dose Analysis of Peptide I treatment in Cancer Patients
[0413] An open-label, multi-center dose escalation study of the
safety, pharmacodynamics, and pharmacokinetics of subcutaneously
administered Peptide I in anemic cancer patients receiving
chemotherapy was conducted. An objective of this study was to
determine the dose of Peptide I administered every three weeks by
subcutaneous (SC) injection associated with a hemoglobin increase
of .gtoreq.1 g/dL at 9 weeks in .gtoreq.50% of anemic cancer
patients receiving chemotherapy. The time frame of the study was
approximately 13 weeks. Results of the study can be summarized, as
shown in Table 13, which groups the results by the three cancer
types studied, breast cancer, non-small cell lung cancer (NSCLC),
and prostate cancer. Further details are discussed below and
presented in Tables 14-16.
TABLE-US-00019 TABLE 13 Cancer Types Breast NSCLC Prostate Hgb No.
of Subjects 8 8 6 No. of Responders 8 4 3 (Hgb Change from BL
.gtoreq. 1 g/dL by Week 9) % Responders 100 50 50 Dose (mg/kg) Mean
0.112 0.088 0.156 Min. 0.050 0.050 0.050 Max. 0.200 0.199 0.212
[0414] Other measures of the study included: (1) an evaluation of
the safety profile of up to four doses of Peptide I administered
subcutaneously every three weeks in cancer patients receiving
concomitant myelosuppressive chemotherapy, (2) a determination of
the change from baseline in hemoglobin (Hgb) in anemic cancer
patients receiving chemotherapy at different dose levels of Peptide
I, (3) a determination of the proportion of patients who have a Hgb
response to Peptide I, (4) a determination of Peptide I
administered subcutaneously that increases and maintains the
hemoglobin in the target range of 11-13 g/dL in anemic cancer
patients receiving chemotherapy, (5) an evaluation of the
pharmacokinetic profile of up to 4 doses of Peptide I administered
subcutaneously in anemic cancer patients receiving chemotherapy (in
a subset of study patients), (6) an exploration of the effect of
dose frequency at an active dose of Peptide I, and (7) and
exploration of the effect of parenteral iron replacement at an
active dose of Peptide I.
[0415] Patient inclusion criteria for this study were as follows:
(1) the Patient was informed of the investigational nature of this
study and gave written, witnessed informed consent in accordance
with institutional, local, and national guidelines; (2) male or
female .gtoreq.18 and .ltoreq.80 years of age; pre-menopausal
females (with the exception of those who are surgically sterile)
must have a negative pregnancy test at screening; those who are
sexually active must practice a highly effective method of birth
control for at least 2 weeks prior to study start, and must be
willing to continue practicing birth control for at least 4 weeks
after the last dose of study drug. A highly effective method of
birth control is defined as one that results in a low failure rate
(i.e., less than 1% per year) when used consistently and correctly
such as implants, injectables, combined oral contraceptives, some
IUDs, sexual abstinence (only acceptable if practiced as a
life-style and not acceptable if one who is sexually active
practices abstinence only for the duration of study) or
vasectomized partner; (3) Patients with histologically confirmed
solid tumor malignancy or lymphoma who are scheduled to receive at
least 9 weeks of cyclic myelosuppressive chemotherapy while on
study; (4) a hemoglobin value of .gtoreq.8 and <11 g/dL within 1
week prior to administration of study drug; (5) an ECOG Performance
Status of 0-2; (6) one reticulocyte hemoglobin content (CHr)>29
pg within 4 weeks prior to study drug administration; (7) one
transferrin saturation .gtoreq.15% within 4 weeks prior to study
drug administration; (8) one serum or red cell folate level above
the lower limit of normal within 4 weeks prior to study drug
administration; (9) one vitamin B.sub.12 level above the lower
limit of normal within 4 weeks prior to study drug administration;
(10) one absolute neutrophil count .gtoreq.1.0.times.10.sup.9/L
within 1 week prior to administration of study drug; (11) one
platelet count .gtoreq.75.times.10.sup.9/L within one week prior to
administration of study drug; and (12) a life expectancy >6
months.
[0416] If the patient was considered by the investigator as
iron-deficient and IV iron supplementation was required, the
patient was re-screened weekly (no sooner than 7 days after iron
administration) until hemoglobin has not increased more than 0.5
g/dL from the previous week.
[0417] Patient exclusion criteria included: (1) treatment with any
erythropoiesis stimulating agent (ESA) in the past 90 days; (2)
history of failure to respond to ESA treatment; (3) known
antibodies to other ESAs or history of pure red cell aplasia
(PRCA); (4) acute or chronic leukemia, myelodysplastic syndrome
(MDS), or multiple myeloma; (5) any previous or planned
radiotherapy to more than 50% of either the pelvis or spine; (6)
known intolerance to parenteral iron supplementation; (7) red-blood
cell (RBC) transfusion within 4 weeks prior to study drug
administration; (8) known hemoglobinopathy (e.g., homozygous
sickle-cell disease, thalassemia of all types, etc.); (9) known
hemolysis; (10) history of pulmonary embolism or DVT in the
previous 2 years or current therapeutic doses of anticoagulants;
(11) known blood loss as a cause of anemia; (12) uncontrolled, or
symptomatic inflammatory disease (e.g., rheumatoid arthritis,
systemic lupus erythematosus, etc.); (13) AST or ALT>2.5 times
the upper limit of normal; AST or ALT>5 times the upper limit of
normal if liver metastases are present; (14) creatinine >175
mmol/L; (15) history of bone marrow or peripheral blood cell
transplantation; (16) pyrexia/fever of .gtoreq.39.degree. C. within
48 hours prior to study drug administration; (17) poorly controlled
hypertension, per the investigator's judgment, within 4 weeks prior
to study drug administration (e.g., systolic .gtoreq.170 mm Hg or
diastolic .gtoreq.100 mm Hg on repeat readings); (18) epileptic
seizure in the 6 months prior to study drug administration;
advanced chronic congestive heart failure--New York Heart
Association Class IV; (19) high likelihood of early withdrawal or
interruption of the study (e.g., myocardial infarction within the
past 3 months; severe or unstable coronary artery disease; stroke;
respiratory, autoimmune, neuropsychiatric or neurological
abnormalities; liver disease including active hepatitis B or C;
active HIV disease; or any other clinically significant medical
diseases or conditions within the prior 6 months that may, in the
investigator's opinion, interfere with assessment or follow-up of
the patient); (20) anticipated elective surgery during the study
period; (21) history of multiple drug allergies; (22) exposure to
any investigational agent within 1 month prior to administration of
study drug or planned receipt during the study period.
[0418] Tables 14-16 provide clinical results by cancer group. The
data includes hemoglobin concentrations (the top portion of each
table) and dosing information (bottom portion of each table) for
Peptide I. The abbreviations used in these tables are as follows:
"Hgb" refers to Hgb concentration in g/dL; "n" indicates the number
of observations; "std" refers to standard deviation, "Taxane"
indicates that the patient was being treated with a taxane-based
chemotherapeutic agent. "Non-Taxane" indicates that the patient was
being treated with a non-taxane based chemotherapeutic agent. Empty
cells indicate that no data was obtained or that no dose was
given.
TABLE-US-00020 TABLE 14 Breast Cancer Patient Group Results.
Treatment Non- Non- Non- Non- Non- Non- Taxane Taxane Taxane Taxane
Taxane Taxane Taxane Taxane Patient ID 1 2 3 4 5 6 7 8 Mean Week
Hgb Hgb Hgb Hgb Hgb Hgb Hgb Hgb Hgb std n -6 10.8 -5 11.9 -4 11.2
-3 10.6 -2 9.7 11.5 9.3 Screen 9.3 10.3 10.8 8.7 9.8 10.8 10.8 9.2
10.1 0.83 8 (-1 week) Dose 1/ 9.4 9.4 10.8 8.6 9.9 10.8 10.6 10.8
9.9 0.84 8 week 1 2 9.5 10.7 8.8 10.9 11.5 11.4 11.2 10.5 1.03 7 3
9.7 10 11.5 8.9 9.8 11.4 12.8 11.7 10.6 1.31 8 Dose 2/ 9.5 9.8 11.9
8.5 10.2 12.1 13.7 12.5 10.8 1.78 8 week 4 5 10.9 9.2 10.4 13.2
14.2 13.4 11.6 1.99 6 6 10.8 11.3 9.7 10.5 14.2 14 14.3 11.8 1.98 7
Dose 3/ 11.6 10.1 9.4 11.3 13.7 13.5 13.4 11.6 1.73 7 week 7 8 7.9
10.1 10.3 11.1 15 13.6 10.9 2.57 6 9 11.7 11.5 10.3 15.7 13.7 12.3
12.6 1.91 6 Dose 4/ 12.6 9.9 11.7 13.9 13.2 11.6 12.3 1.41 6 week
10 11 12.5 11 11.7 15.6 13.2 11.6 12.8 1.66 6 12 13.1 10.1 12 14.6
13.4 12.1 12.6 1.53 6 13 12.9 10.9 14.7 13.1 11.5 12.9 1.49 5 14
13.3 10.4 12.9 11.8 12.2 1.30 4 15 10.1 13.7 12.4 12.8 12.1 1.53 4
16 12.1 12.6 13.5 12.5 17 13.3 18 12.9 19 12.1 20 12.3 21 12.9 22
12.7 23 12.4 Baseline 9.35 9.85 10.8 8.65 9.85 10.8 10.7 10 Hgb yes
yes yes yes yes yes yes yes Change .gtoreq. 1 % responders 100 Dose
Summary (calculated dose, mg/kg) Week 1 0.15 0.1 0.05 0.1 0.148
0.148 0.2 0.2 Week 4 0.15 0.1 0.1 0.148 0.148 0.1 0.188 Week 7 0.15
0.1 0.1 0.148 0.2 0.094 Week 10 0.15 0.1 0.148 0.1 0.1 Total # Dose
4 4 1 3 4 2 4 4 Mean 0.112 min 0.05 Max 0.2
TABLE-US-00021 TABLE 15 Non-Small Cell Lung Cancer Patient Group
Results. Treatment Non- Non- Non- Non- Non- Non- Non- Non- Taxane
Taxane Taxane Taxane Taxane Taxane Taxane Taxane Patient ID 9 10 11
12 13 14 15 16 Week Hgb Hgb Hgb Hgb Hgb Hgb Hgb Hgb Mean Std n -6
-5 -4 10.3 -3 8.9 9.8 -2 9.6 8.7 Screen 9.1 9.8 10.8 10.8 9.3 10.7
10.3 7.9 10.1 1.03 8 (-1 week) Dose 1/ 8.9 10.1 10.8 12.3 9.5 9.8
10.8 8 10.3 1.31 8 week 1 2 9.9 9.3 10 11.6 9.8 10.8 8.6 10.2 0.97
7 3 8.7 9.8 10.3 8.4 11.9 6.6 9.8 1.81 6 Dose 2/ 9.6 10 11.4 10.9
11.3 9.4 6.7 10.4 1.62 7 week 4 5 10.7 9.3 12 11.2 9.4 9.2 10.5
1.17 6 6 10.7 10.6 10.3 11.3 8.3 9.8 10.2 1.04 6 Dose 3/ 11 9.7
11.9 9.6 9.4 10.6 1.08 5 week 7 8 13.1 10.5 11.5 11.8 9.4 10.2 11.3
1.32 6 9 14.1 10.5 11.5 11.7 9.3 10.3 11.4 1.65 6 Dose 4/ 10.8 9.7
12.4 8.9 10.5 1.52 4 week 10 11 9.7 11.5 6.9 9.4 2.32 3 12 11.3
10.8 11.6 6.1 11.1 10.0 2.30 5 13 10.8 10.6 11.6 11 11.0 0.43 4 14
11.6 10.4 11.7 10.5 10.7 1.10 5 15 11.2 11.2 1 16 17 Baseline 9
9.95 10.8 11.55 9.4 10.25 10.55 7.95 Hgb Change .gtoreq. 1 yes no
no no yes no yes yes % responders 50 Dose Summary (calculated dose,
mg/kg) Week 1 0.199 0.156 0.053 0.05 0.1 0.05 0.05 0.05 Week 4
0.199 0.156 0.053 0.05 0.1 0.05 0.05 Week 7 0.197 0.053 0.05 0.1
0.05 0.05 Week 10 0.053 0.05 0.1 0.05 0.05 Total # Dose 3 2 4 4 4 4
1 4 Mean 0.08825 min 0.05 max 0.199
TABLE-US-00022 TABLE 16 Non-Small Cell Lung Cancer Patient Group
Results. Treatment Non- Taxane Taxane Taxane Taxane Taxane Taxane
Patient ID 17 18 19 20 21 22 Week Hgb Hgb Hgb Hgb Hgb Hgb Mean Std
n -6 -5 -4 -3 -2 Screen 10.3 10.2 10.3 10.6 9.7 9.7 10.2 0.36 6 (-1
week) Dose 1/ 10.5 10 10.1 10.5 9.5 10.3 10.1 0.38 6 week 1 2 11.8
10.1 10.1 11 8.8 11.1 1.33 6 3 9.9 10.1 10.7 8.8 10.6 1.00 5 Dose
2/ 12.6 10 10.6 11.5 8.9 11.1 1.27 6 week 4 5 10.5 12.2 11.7 8.7
11.5 1.56 4 6 10.1 10.5 10.4 8.7 10.3 0.83 4 Dose 3/ 11.9 10.3 10.9
12 9.8 8.9 11.0 1.21 6 week 7 8 11.2 12.7 13.1 9.4 9.1 11.6 1.83 5
9 9.9 11 9.3 8 10.1 1.25 4 Dose 4/ 10.9 13.8 8.1 10.9 2.85 3 week
10 11 10 10.9 13.7 7.6 10.6 2.52 4 12 9.7 11.4 13 11.4 1.56 4 13
9.4 11.2 10.3 1.27 2 14 9.8 11.2 10.5 0.99 2 15 12.9 11.0 2.69 2 16
Baseline 10.4 10.1 10.2 10.55 9.6 10 Hgb yes no yes yes no no
Change .gtoreq. 1 % responders 50 Dose Summary (calculated dose,
mg/kg) Week 1 0.05 0.15 0.15 0.204 0.193 0.199 Week 4 0.15 0.15
0.212 0.187 0.199 Week 7 0.15 0.141 0.204 0.193 0.199 Week 10 0.15
0.141 0.204 0.193 Total # Dose 1 4 4 4 4 3 Mean 0.156167 min 0.05
max 0.212
[0419] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0420] It is further to be understood that all values are
approximate, and are provided for description.
[0421] Numerous references, including patents, patent applications,
and various publications are cited and discussed in the description
of this invention. The citation and/or discussion of such
references is provided merely to clarify the description of the
present invention and is not an admission that any such reference
is "prior art" to the present invention. All references cited and
discussed in this specification are incorporated herein by
reference in their entirety and to the same extent as if each
reference was individually incorporated by reference.
Sequence CWU 1
1
14120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr
Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Lys 20 221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10
15 Pro Leu Arg Gly Lys 20 320PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr
Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20 55PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Gly
Gly Leu Tyr Ala 1 5 64PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Cys His Met Gly 1
78PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Pro Ile Thr Xaa Val Cys Gln Pro 1 5
83PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Leu Arg Gly 1 99PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Gly
Gly Leu Tyr Ala Cys His Met Gly 1 5 1011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Pro
Ile Thr Xaa Val Cys Gln Pro Leu Arg Gly 1 5 10 1120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10
15 Pro Leu Arg Gly 20 1220PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
1320PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr
Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20 1419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Ala Val Cys Gln 1 5 10
15Pro Leu Arg
* * * * *