Note: Descriptions are shown in the official language in which they were submitted.
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MODIFIED GLP-1 RECEPTOR AGONISTS AND
THEIR PHARMACOLOGICAL METHODS OF USE
[001] This application claims benefit of U.S. Provisional Application Serial
No.
60/408,696, filed September 6, 2002, and U.S. Provisional Application Serial
No.
601439,369, filed January 9, 2003, the contents of which are incorporated
herein by
reference in their entirety.
FIELD OF THE INVENTION
[002] This invention relates to modified GLP-1 receptor agonists comprising a
GLP-1
receptor agonist linked to a polyethylene glycol polymer having a molecular
weight of
greater than 30 kD, as well as related formulations, dosages and methods of
administration thereof for therapeutic purposes. More particularly, these
modified GLP-1
receptor agonists, compositions and methods are useful in providing a
treatment option
for those individuals afflicted with a metabolic disorder such as diabetes,
impaired
glucose tolerance, or metabolic syndrome, prediabetic states, by inducing
glucose-
dependent insulin secretion in the absence of the therapeutically limiting
side effect of
reducing or inhibiting gastrointestinal motility.
BACKGROUND OF THE RELATED ART
[003] Diabetes is characterized by impaired insulin secretion manifesting
itself, among
other things, by an elevated blood glucose level in the diabetic patient.
Underlying
defects lead to a classification of diabetes into two major groups: type 1
diabetes, or
insulin dependent diabetes mellitus (IDDM), which arises when patients lack (3-
cells
producing insulin in their pancreatic glands, and type 2 diabetes, or non-
insulin
dependent diabetes mellitus (NIDDM), which occurs in patients with an impaired
[3-cell
insulin secretion and alterations in insulin action.
[004] Type 1 diabetic patients are currently treated with insulin, while type
2 diabetic
patients can be treated with agents that stimulate (3-cell function or with
agents that
enhance the tissue sensitivity of the patients towards insulin. Over time
almost one-half
of type 2 diabetic subjects lose their response to these agents and then must
be placed
on insulin therapy. The drugs presently used to treat type 2 diabetes are
described
below.
[005] Alpha-glucosidase inhibitors (e.g., PRECOSE~, VOGLIBOSETM, and
MIGLITOL~)
reduce the excursion of postprandial glucose by delaying the absorption of
glucose from
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the gut. These drugs are safe and provide treatment for mild to moderately
affected
diabetic subjects. However, gastrointestinal side effects have been reported
in the
literature and limit their effectiveness.
[006] Insulin sensitizers are drugs that enhance the body's response to
insulin.
Thiozolidinediones such as AvandiaTM (rosiglitazone) and ActosTM activate the
Peroxisome proliferator-activated receptor (PPAR) gamma and modulate the
activity of a
set of genes that have not been well described. RezulinTM (troglitazone), the
first drug in
this class, was withdrawn because elevated liver enzyme levels and drug
induced
hepatotoxicity. These hepatic effects do not appear to be a significant
problem in patients
using AvandiaTM and ActosTM. Even so, liver enzyme testing is recommended
every 2
months in the first year of therapy and periodically thereafter. AvandiaTM and
ActosTM
treatments are associated with fluid retention, edema and weight gain.
AvandiaTM is not
indicated for use with insulin because of concern about congestive heart
failure.
[007] Insulin secretagogues such as sulfonylureas (SFUs) and the non-
sulfonylureas
(e.g., Nateglinide and Pepaglinide) act through the ATP-dependent K+ channel
to cause
glucose independent insulin secretion. These drugs are standard therapy for
type 2
diabetics that have mild to moderate fasting glycemia. The SFUs have
limitations that
include a potential for inducing hypoglycemia, weight gain, and high primary
and
secondary failure rates. Ten to 20% of initially treated patients fail to show
a significant
treatment effect (primary failure). Secondary failure is demonstrated by an
additional 20-
30% loss of treatment effect after six months on an SFU. Insulin treatment is
required in
50% of the SFU responders after 5-7 years of therapy (Scheen, et al., Diabetes
Res. Clin.
Pract. 6:533-543, 1989). Nateglinide and Pepaglinide are short-acting drugs
that need to
be taken three times a day. They are used only for the control of post-
prandial glucose
and not for control of fasting glucose.
[008] GLUCOPHAGETM (metformin HCI) is a biguanide that lowers blood glucose by
decreasing hepatic glucose output and increasing peripheral glucose uptake and
utilization. The drug is effective at lowering blood glucose in mildly and
moderately
affected subjects, and does not have the side effects of weight gain or the
potential to
induce hypoglycemia. However, GLUCOPHAGETM has a number of side effects
including
gastrointestinal disturbances and lactic acidosis. GLUCOPHAGETM is
contraindicated in
diabetics over the age of 70 and in subjects with impairment in renal or liver
function.
Finally, GLUCOPHAGETM has the same primary and secondary failure rates as the
SFUs.
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[009] Insulin treatment is instituted after diet, exercise, and oral
medications have failed
to adequately control blood glucose. This treatment has the drawbacks that it
is an
injectable, can produce hypoglycemia, and causes weight gain. The possibility
of
inducing hypoglycemia with insulin limits the extent that hyperglycemia can be
controlled.
[010] Because of the problems with current treatments, new therapies to treat
type 2
diabetes are needed. In particular, new treatments to retain normal (glucose-
dependent)
insulin secretion are needed. Such new drugs should have the following
characteristics:
dependent on glucose for promoting insulin secretion (i.e., produce insulin
secretion only
in the presence of elevated blood glucose); low primary and secondary failure
rates; and
preserve islet cell function. The strategy to develop the new therapy
disclosed herein is
based on the cyclic adenosine monophosphate (CAMP) signaling mechanism and its
effects on insulin secretion.
[011] Glucose is a major regulator of the insulin secretion process. Elevation
of this
sugar promotes the closure of the K+ channels following the elevation of ATP.
Closure of
the K+ channels causes cell depolarization and subsequent opening of Ca++
channels,
which in turn leads to exocytosis of insulin granules. Little, if any, effects
on insulin
secretion occurs in the absence of low glucose concentrations (Weinhaus, et
al.,
Diabetes 47:1426-1435, 1998). Secretagogues like GLP-1 utilize the cAMP system
to
regulate insulin secretion through this glucose-dependent mechanism (Komatsu,
et al.,
Diabetes 46:1928-1938, 1997; Filipsson, et al., Diabetes 50:1959-1969, 2001;
Drucker,
Endocrinology 142:521-527, 2001 ). Insulin secretagogues via the elevation of
cAMP is
also able to enhance insulin synthesis in addition to insulin release
(Skoglund, et al.,
Diabetes 49:1156-1164, 2000; Borboni, et al., Endocrinology 140:5530-5537,
1999).
[012] GLP-1 (glucagon-like peptide 1 ) is released from the intestinal L-cell
after a meal
and functions as an incretin hormone (i.e., it potentiates glucose-induced
insulin release
from the pancreatic ~i-cell). It is a 37-amino acid peptide that is
differentially expressed by
the glucagon gene, depending upon tissue type. The clinical data that support
the
beneficial effect of raising cAMP levels in (3-cells have been collected with
GLP-1.
Infusions of GLP-1 in poorly controlled type 2 diabetics normalized their
fasting blood
glucose levels (Gutniak, et al., New Eng. J. Med. 326:1316-1322, 1992) and
with longer
infusions improved the (3-cell function to those of normal subjects (Rachman,
et al.,
Diabetes 45:1524-1530, 1996). A recent report has shown that GLP-1 improves
the
ability of ~i-cells to respond to glucose in subjects with impaired glucose
tolerance (Byrne,
et al., Diabetes 47:1259-1265, 1998). All of these effects, however, are short-
lived
because of the short half-life of the peptide.
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[013] In addition to the short half-life of the GLP-1 peptide, GLP-1 reduces
gut motility
(see, e.g., Kieffer and Habener, Endocrine Reviews 20:876-913, 1999 and
Drucker,
Gastroenterology 122:531-544, 2002), which in turn results in significant
gastrointestinal
side effects, such as nausea and vomiting. Such gastrointestinal side effects
have also
been demonstrated with GLP-1 agonists, such as NN-2211 (Agerso, et al.,
Diabetologia
45:195-202, 2002) and Exendin-4 (Amylin abstract, American Diabetes
Association
meetin, 2001 ). The reduction in gut motility that causes the gastrointestinal
side effects
has been studied in rodent models (Lotti, et al., Life Sci. 39:1631-1638,
1986). The
inhibitory effects of GLP-1 on the gastrointetestinal motility and secretion
have been
shown to be at least partially mediated by the central nervous system
(Imeryuz, et.al.,
Am. J. Physiol 273:6920-6927, 1997). Systemically injected GLP-1 gains access
to the
brain from the periphery by simple diffusion (Kastin, et al., J. Mol.
Neurosci. 18:7-14,
2002).
[014] Therefore, there exists a need for improved peptides that have the
glucose-
dependent insulin secretagogue activity of GLP-1, but without the side effects
which limits
GLP-1 based treatments.
SUMMARY OF THE INVENTION
[015] This invention relates to modified GLP-1 receptor agonists comprising a
GLP-1
receptor agonist linked to a polyethylene glycol polymer having a molecular
weight of
greater than 30 kD, and which retains its ability to agonize the GLP-1
receptor. These
modified GLP-1 agonists are effective in the treatment of metabolic disorders,
such as
diabetes or impaired glucose tolerance, a prediabetic state. Moreover, the
modified GLP-
1 agonists of this invention are capable of treating metabolic disorders
without inhibiting
gastrointestinal motility, thereby producing fewer gastrointestinal side
effects, such as
nausea and vomiting and allowing higher more effective doses to be
administered.
[016] In particular, one aspect of the invention is a polypeptide that
functions as a GLP-1
receptor agonist. Examples of GLP-1 receptor agonists include, but are not
limited to, the
polypeptides shown in Figure 1 and include those polypeptides selected from
the group
consisting of SEQ ID NOs: 1-10, and fragments and variants of the polypeptide
that
function as an agonist of the GLP-1 receptor at substantially the same level
as the
polypeptides of the listed SEQ ID NOs: 1-10 (collectively, polypeptides of
this invention).
[017] Another embodiment of the invention is polynucleotides that encode for
the GLP-1
receptor agonist polypeptides, and the attendant vectors and host cells
necessary to
recombinantly express the polypeptides of this invention.
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[018] Another aspect of this invention is a modified GLP-1 receptor agonist
comprising
one of the polypeptides of this invention, or fragments or variants thereof,
that function as
an agonist of the GLP-1 receptor at substantially the same level as the
polypeptides of
the invention that has been modified by linking to it a polyethylene glycol
polymer having
a molecular weight of greater than 30 kD (collectively, "modified polypeptides
of this
invention"). Examples of modified GLP-1 receptor agonists include, but are not
limited
to, the modified polypeptides shown in Figure 2 and include those polypeptides
selected
from the group consisting of SEQ ID NOs: 13-14 and 16-31.
[019] The invention is also directed to methods of making the GLP-1 agonist
polypeptides of this invention, both recombinant and synthetic, and methods of
making
the modified GLP-1 agonist polypeptides of this invention.
[020] Also disclosed are methods of treating diabetes andlor other diseases or
conditions in a mammal without causing gastrointestinal side effects,
comprising
administering a therapeutically effective amount of any of the modified
polypeptides of the
present invention to said mammal.
BRIEF DESCRIPTION OF THE DRAWING
[021] FIG. 1 depicts amino acid sequences of polypeptides identified as SEQ ID
NOs:1-
which are examples of GLP-1 receptor agonists, the polypeptides of the
invention.
[022] FIG. 2 depicts amino acid sequences of polypeptides of SEQ ID NOs:13-14
and
16-31, which are examples of modified GLP-1 agonists, the modified
polypeptides of this
invention.
[023] FIG. 3A is a line graph showing that fatty acid-modified GLP-1 reduced
gastrointestinal motility.
[024] FIG. 3B is a line graph showing that GLP-1 modified with a 22 kD PEG
reduced
gastrointestinal motility, whereas GLP-1 modified with a 43 kD PEG did not
reduce
gastrointestinal motility.
[025] FIG. 4 is a line graph showing that a GLP-1 agonist of this invention
(SEQ ID NO:
26) modified with a 22 kD or 30 kD PEG reduced gastrointestinal motility,
whereas the
same GLP-1 agoinst peptide modified with a 43 kD PEG did not reduce
gastrointestinal
motility.
[026] FIG. 5 is a bar graph showing that a GLP-1 agonist of this invention
(SEQ ID NO:
26) modified with a 43 kD PEG will reduce gastrointestinal motility if
injected ICV.
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DESCRIPTION OF THE INVENTION
[027] This invention relates to modified GLP-1 receptor agonists comprising a
GLP-1
receptor agonist linked to a polyethylene glycol (PEG) polymer having a
molecular weight
of greater than 30 kD, and methods of administration thereof for therapeutic
purposes are
provided. More particularly, these modified GLP-1 receptor agonists and
compositions
function in vivo as GLP-1 receptor agonists in the prevention and/or treatment
of such
diseases or conditions as diabetes, hyperglycemia, impaired glucose tolerance,
impaired
fasting glucose, and obesity by inducing glucose-dependent insulin secretion,
without
reducing gastrointestinal motility.
[028] Peptides having GLP-1 receptor agonist activity have been identified,
and include,
for example, GLP-1 (7-36), GLP-1 (7-37), Exendin-4, and other GLP-1 analogs
(see, e.g.,
WO 98/43658, WO 00115224, WO 00/16797, WO 01/98331, U.S. Patent No. 5,545,618;
U.S. Patent No. 5,118,666; and U.S. Patent Application Serial No. 60/395,738;
the
references of which are incorporated herein in their entirety).
[029] The stacking alignment below shows the primary structural relationships
between
some of the known GLP-1 agonist peptides:
SEQ
ID NO.
GLP-1 (7-36) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 1
GLP-1 (7-37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 2
Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 3
(G27) HSQGTFTSDYAKYLDARRAKEFIAWLVKGR-NH2 4
[030] As used herein, the single letter abbreviation for a particular amino
acid, its
corresponding amino acid, and three letter abbreviation are as follows: A,
alanine (ala); C,
cysteine (cys); D, aspartic acid (asp); E, glutamic acid (glu); F,
phenylalanine (phe); G,
glycine (gly); H, histidine (his); I, isoleucine (ile); K, lycine (lys); L,
leucine (leu); M,
methionine (met); N, asparagine (asn); P, proline (pro); Q, glutamine (gln);
R, arginine
(arg); S, serine (ser); T, threonine (thr); V, valine (val); W, tryptophan
(trp); and Y, tyrosine
(tyr).
[031] These polypeptides play a role in glucose homeostatsis, and in
particular, these
peptides function as GLP-1 receptor agonists by lowering plasma glucose
concentrations.
Given GLP-1's role in promoting glucose-regulated insulin secretion in the
pancreas,
GLP-1 receptor agonists are potentially valuable in the treatment of metabolic
disorders
and other diseases. To date, however, GLP-1 receptor agonists have had
significant
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side effects; namely a reduction in gastrointestinal motility, which in turn
can lead to
nausea and vomiting.
[032] It is well known in the art that PEGylation of a drug such as small
molecules,
peptides, or proteins can improve plasma half-life, physical solubility and
stability, and
resistance to protease degradation as well as reduce immunogenicity.
Furthermore, it
has been suggested in the art that PEGylation can reduce the extent of adverse
side
effects by reducing the trough to peak levels of the drug that result from
sustained plasma
concentrations. However, it was not known in the art that PEGylation could
limit access
of a drug to a certain tissue.
[033] Suprisingly, modification of a peptide or protein with a particular size
or structure of
a polymer, such as PEG, can selectively affect tissue distribution of the
administered
modified peptide or protein. For example, modification of GLP-1 agonists with
a linear
22 kD PEG does not increase the therapeutic index (glucose-lowering vs. gut
motility) as
compared to unmodified GLP-1 agonists and C16-fatty acid modified agonists.
Modification with a linear 30 kD PEG modestly improved the therapeutic index,
whereas
modification with a branched 43 kD PEG greatly reduced CNS-mediated gut
motility.
Interestingly, when injected directly into the brain, the 43 kD-PEGylated GLP-
1 agonist
was able to induce gut motility. Thus, PEG size and structure are major
determinants of
such a selective process.
[034] Despite the many advantages attributed to PEGylation, one significant
disadvantage is the interference of the bulky PEG in the interaction between
the peptide
and its receptor, resulting in a reduction of functional activity. Utilizing
GLP-1 and
glucagon as models, a "twisted helix" model was developed, and based on this
model,
positions located within the C-terminal of the peptide were selected for
PEGylation.
These positions were predicted to be on the opposing side of the peptide-
receptor
interaction.
[035] The inventors herein have found that modifying the GLP-1 receptor
agonists by
linking a polyethylene glycol polymer having a molecular weight of greater
than 30 kD to
the GLP-1 receptor agonist will inhibit the reduction in gastrointestinal
motility typically
associated with GLP-1 receptor agonists. Without being bound to theory, the
inventors
herein believe that increasing the size of the GLP-1 receptor agonist using
PEGylation
technology prevents the GLP-1 receptor agonist from crossing the blood-brain-
barrier and
thus, accessing the central nervous system. As a result, the GLP-1 agonists'
ability to
cause gastrointestinal side effects (which are likely to be mediated by the
central nervous
system) is reduced. The PEGylated GLP-1 receptor agonist, however, still has
access to
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the pancreas and thus, lowers blood glucose, the desired activity for treating
type 2
diabetes.
[036] GLP-1 Receptor Aaonists:
The polypeptides of this invention are GLP-1 receptor agonists and are
determined as
such by their ability to activate the GLP-1 receptor. The GLP-1 receptor
agonist activates
the GLP-1 receptor in one or more in vitro or in vivo assays for GLP-1
receptor activation.
Examples of such assays include, but are not limited to, in vitro assays for
induction of
cAMP in RINmSF cells, in vitro assays for induction of insulin secretion from
pancreatic (3-
cells, in vivo assays for reduction in plasma glucose levels, and in vivo
assays for
elevation in plasma insulin levels as described in the specific examples
below.
[037] Examples of the GLP-1 receptor agonists include, but are not limited to,
the
polypeptides selected from the group consisting of SEQ ID NOs: 1-10 and
fragments,
derivatives, variants and analogs thereof, that function as an agonist of the
GLP-1
receptor at substantially the same level as the polypeptides of the listed SEQ
ID NOs:
1-10
[038] GLP-1 receptor agonists, the polypeptides of the present invention, may
be
naturally-occurring polypeptides, recombinant polypeptides, or synthetic
peptides.
[039] Fragments derivatives variants and analogs of GLP-1 receptor aaonists:
Fragment, derivative, variant, and analog polypeptides retain substantially
the same
biological function or activity as, for example, polypeptides shown in SEQ ID
NOS: 1-10.
"Substantially the same biological function or activity" each means that
degree of
biological activity that is within about 30% to about 100% (i.e., 30, 40, 50,
60, 70, 80, 90,
or 100%) or more of that biological activity demonstrated by the polypeptide
to which it is
being compared when the biological activity of each polypeptide is determined
by the
same procedure.
[040] A fragment is less than a full-length polypeptide, such as the
polypeptides shown in
SEQ ID NOs: 1-10, which retains substantially similar functional activity, as
shown in the
in vitro and in vivo models disclosed herein.
[041] Derivatives include polypeptides that have been chemically modified to
provide an
additional structure and/or function. For example, a fatty acid can be added
to a
polypeptide to improve its half-life. Fusion polypeptides which confer
targeting specificity
or an additional activity also can be constructed, as described in more detail
below.
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[042] Derivatives can be modified by either a natural process, such as
posttranslational
processing, or by chemical modification techniques, both of which are well
known in the
art. Modifications can occur anywhere in a polypeptide, including the peptide
backbone,
the amino acid side-chains, and the amino or carboxyl termini. It will be
appreciated that
the same type of modification may be present to the same or varying degrees at
several
sites in a given polypeptide. Also, a variant may contain one or more
different types of
modifications. Polypeptides may be branched, for example, as a result of
ubiquitination,
and they may be cyclic, with or without branching.
[043] Other chemical modifications include acetylation, acylation, ADP-
ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme
moiety, covalent
attachment of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid
derivative, covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide
bond formation, demethylation, formation of covalent cross-links, formation of
cysteine,
formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation,
GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to proteins such as
arginylation,
and ubiquitination (see, e.g., T. E. Creighton, PROTEINS, STRUCTURE AND
MOLECULAR
PROPERTIES, 2nd ed., W.H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, ed.,
Academic Press, New York, pgs. 1-14 (1983); Seifter, et al., Meth. Enzymol
182:626-46,
1990; Rattan, et al., Ann. N.Y. Acad. Sci. 663:48-62, 1992).
[044] Derivatives also include mature polypeptides that have been fused with
another
polypeptide, for example, human serum albumin, to improve their
pharmacokinetic profile.
Fusion of two polypeptides can be accomplished by any means known to one
skilled in
the art. For example, a DNA encoding human serum albumin and a DNA sequence
encoding a polypeptide of the invention can be cloned into any mammalian
expression
vector known to one skilled in the art. Location of a polypeptide of the
invention N-
terminal to the other polypeptide is preferred, because it appears that a free
N-terminal
histidine is required for GLP-1 receptor activity (Kawa, Endocrinology
124(49):1768-73,
1989). The resulting recombinant fusion protein can then be expressed by
transforming a
suitable cell line, such as HKB or CHO, with the vector and expressing the
fusion protein.
[045] Variants of polypeptides of the invention include polypeptides having
one or more
amino acid sequence changes with respect to the amino acid sequences shown in
SEQ
ID NOS: 1-10. Variants also can have amino acids joined to each other by
modified
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peptide bonds (i.e., peptide isosteres) and may contain amino acids other than
the 20
naturally occurring amino acids.
[046] Preferably, variants contain one or more conservative amino acid
substitutions
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions), preferably at
nonessential amino acid
residues. A "nonessential" amino acid residue is a residue that can be altered
from a
wild-type sequence of a protein without altering its biological activity,
whereas an
"essential" amino acid residue is required for biological activity. A
conservative amino
acid substitution is one in which the amino acid residue is replaced with an
amino acid
residue having a similar side chain. Families of amino acid residues having
similar side
chains have been defined in the art. These families include amino acids with
basic side
chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
(e.g., threonine, valine, isoleucine), and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Non-conservative substitutions would
not be made
for conserved amino acid residues or for amino acid residues residing within a
conserved
protein domain.
[047] Variants also include polypeptides that differ in amino acid sequence
due to
mutagenesis. Variants that function as GLP-1 receptor agonists can be
identified by
screening combinatorial libraries of mutants, for example, mutants of
polypeptides with
conservative substitutions at one or more positions (i.e., at 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10
positions) can be screened for GLP-1 receptor agonist activity using methods
well known
in the art, and described in Examples 3, 4, 5, and 6.
[048] An analog includes a propolypeptide, which includes an amino acid
sequence of a
polypeptide of the invention. Active polypeptides of the invention can be
cleaved from the
additional amino acids in the propolypeptide molecule by natural, in vivo
processes, or by
procedures well known in the art, such as by enzymatic or chemical cleavage.
[049] Polynucleotides of this Invention and Methods of Producing the
Polypeptides
Any polynucleotide sequence that encodes a polypeptide of the invention can be
used to
express the polypeptide. Polynucleotides can consist only of a coding sequence
for a
polypeptide or can include additional coding and/or non-coding sequences.
[050] Polynucleotide sequences encoding a polypeptide of the invention can be
synthesized in whole or in part using chemical methods well known in the art
(see, e.g.,
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Caruthers, et al., Nucl. Acids Res. Symp. Ser. 215-23, 1980; Horn, et al.,
Nucl. Acids
Res. Symp. Ser 225-32, 1980). The polynucleotide that encodes the polypeptide
can
then be cloned into an expression vector to express the polypeptide, or into a
cloning
vector to propagate the polynucleotide.
[051] As will be understood by those of skill in the art, it may be
advantageous to
produce the polypeptide-encoding nucleotide sequences possessing non-naturally
occurring codons. For example, codons preferred by a particular prokaryotic or
eukaryotic host can be selected to increase the rate of polypeptide expression
or to
produce an RNA transcript having desirable properties, such as a half-life,
which is longer
than that of a transcript generated from the naturally occurring sequence.
[052] The nucleotide sequences disclosed herein can be engineered using
methods
generally known in the art to alter the polypeptide-encoding sequences for a
variety of
reasons, including but not limited to, alterations which modify the cloning,
processing,
and/or expression of the polypeptide or mRNA product. DNA shuffling by random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides can
be used to engineer the nucleotide sequences. For example, site-directed
mutagenesis
can be used to insert new restriction sites, alter glycosylation patterns,
change codon
preference, produce splice variants, introduce mutations, and so forth.
[053] The present invention also includes cloning and expression vectors
comprising one
or more nucleotide sequences encoding a polypeptide of the invention. The
nucleotide
sequence can be inserted in a forward or reverse orientation. A DNA sequence
may be
inserted into a vector by a variety of procedures. In general, a DNA sequence
is inserted
into an appropriate restriction endonuclease site by procedures known in the
art and
described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed.,
(Cold Spring Harbor, N.Y., 1989). Such procedures and others are deemed to be
within
the scope of those skilled in the art.
[054] Examples of cloning vectors include, but are not limited to pBR322,
pUC18,
pUC19, pSport, and pCRll.
[055] An expression vector may further comprise regulatory sequences,
including, for
example, a promoter, operably linked to the coding sequence. Large numbers of
suitable
expression vectors and promoters are known to those of skill in the art and
are
commercially available. The following expression vectors are provided by way
of
example. Bacterial expression vectors include, but are not limited to, pQE70,
pQE60,
pQE-9 (Qiagen); pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNHBa,
pNH16a,
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pNH18a, pNH46a (Stratagene); and pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5
(Pharmacia). Eukaryotic expression vectors include, but are not limited to,
pWLneo,
pSV2cat, pOG44, pXT1, pSG (Stratagene); and pSVK3, pBPV, pMSG, PSVL
(Pharmacia). However, any other cloning or expression vector may be used as
long as it
is replicable and viable in the desired host. Promoter regions can be selected
from any
desired gene using CAT (chloramphenicol transferase) expression vectors or
other
vectors with selectable markers. Two appropriate vectors are pKK232-8 and
pCM7.
Particular named bacterial promoters include laci, IacZ, T3, T7, gpt, lambda
PR, P~ and
trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,
early
and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of
an
appropriate vector and promoter is well within the level of ordinary skill in
the art.
[056] An expression vector also may contain a ribosome binding site for
translation
initiation, a transcription terminator, and appropriate sequences for
amplifying expression.
Expression vectors may contain a gene to provide a phenotypic trait for
selection of
transformed host cells, such as dihydrofolate reductase or neomycin resistance
for a
eukaryotic cell culture, or such as tetracycline or ampicillin resistance for
culture in E. coli.
[057] In one embodiment, a library of variants is generated by combinatorial
mutagenesis at the nucleic acid level and is encoded by a variegated gene
library. A
library of variants can be produced, for example, by enzymatically ligating a
mixture of
synthetic oligonucleotides into gene sequences such that a degenerate set of
potential
variant amino acid sequences is expressible as individual polypeptides or,
alternatively,
as a set of larger fusion proteins (e.g., for phage display) containing the
set of sequences
therein.
[058] There are a variety of methods that can be used to produce libraries of
potential
variants from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA synthesizer, and
the
synthetic gene then ligated into an appropriate expression vector. Use of a
degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding the
desired set of potential analog sequences. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang, Tetrahedron 39:3,
1983; Itakura,
et al., Annu. Rev. Biochem. 53:323, 1984; Itakura, et al., Science 198:1056,
1984; Ike, et
al., Nucleic Acid Res. 11:477, 1983).
[059] Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
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rapid screening of the gene libraries generated by the combinatorial
mutagenesis of
polypeptides of the invention. The most widely used techniques, which are
amenable to
high through-put analysis for screening large gene libraries, typically
include cloning the
gene library into replicable expression vectors, transforming appropriate
cells with the
resulting library of vectors, and expressing the combinatorial genes under
conditions in
which detection of a desired activity facilitates isolation of the vector
encoding the gene
whose product was detected. Recursive ensemble mutagenesis (REM), a technique
that
enhances the frequency of functional mutants in the libraries, can be used in
combination
with the screening assays to identify the desired variants.
[060] The present invention also provides host cells containing the above-
described
vectors. The host cell can be a higher eukaryotic cell, such as a mammalian
cell, or a
lower eukaryotic cell, such as a yeast cell. Alternatively, the host cell can
be a
prokaryotic cell, such as a bacterial cell.
[061] Host cells can be genetically engineered (transduced, transformed, or
transfected)
with cloning or expression vectors of the invention. The vector may be, for
example, in
the form of a plasmid, a viral particle, or a phage. Engineered host cells can
be cultured
in conventional nutrient media modified as appropriate for activating
promoters or
selecting transformants. The selection of appropriate culture conditions, such
as
temperature and pH, are well within the skill of the ordinarily skilled
artisan.
[062] As representative examples of appropriate hosts, include, but are not
limited to,
bacterial cells such as E. coli, Salmonella typhimurium, or Streptomyces;
fungal cells
such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; or
mammalian
cells such as CHO, COS, or Bowes melanoma. The selection of an appropriate
host is
deemed to be within the scope of those skilled in the art from the teachings
herein.
[063] Introduction of the construct into the host cell can be efFected, for
example, by
calcium phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation
(Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, 1936). Constructs in host
cells
can be used in a conventional manner to produce the gene product encoded by
the
recombinant sequence.
[064] Mature proteins can be expressed in mammalian cells, yeast, bacteria, or
other
cells under the control of appropriate promoters. Cell-free translation
systems can also
be employed to produce such proteins using RNAs derived from the DNA
constructs of
the present invention. Appropriate cloning and expression vectors for use with
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prokaryotic and eukaryotic hosts are described above and in Sambrook, et al.,
(MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., Cold Spring Harbor, N.Y.,
1989).
[065] Transcription of a DNA encoding polypeptides of the present invention by
higher
eukaryotes can be increased by inserting an enhancer sequence into the
expression
vector. Enhancers are cis-acting elements of DNA, usually from about 10 to 300
bp, that
act on a promoter to increase its transcription. Examples include the SV40
enhancer on
the (ate side of the replication origin (bp 100 to 270), a cytomegalovirus
early promoter
enhancer, a polyoma enhancer on the late side of the replication origin, and
adenovirus
enhancers. Generally, recombinant expression vectors will include origins of
replication
and selectable markers permitting transformation of the host cell (e.g., the
ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 gene) and a promoter derived
from a
highly expressed gene to direct transcription of a downstream structural
sequence. Such
promoters can be derived from operons encoding glycolytic enzymes such as 3-
phosphoglycerate kinase (PGK), a factor, acid phosphatase, or heat shock
proteins,
among others. The heterologous structural sequence is assembled in appropriate
phase
with translation, initiation and termination sequences, and preferably, a
leader sequence
capable of directing secretion of translated protein into the periplasmic
space or
extracellular medium. Optionally, the heterologous sequence can encode a
fusion protein
including an N-terminal identification peptide imparting desired
characteristics (e.g.,
stabilization or simplified purification of expressed recombinant product).
[066] After transformation of a suitable host strain and growth of the host
strain to an
appropriate cell density, the selected promoter is derepressed by appropriate
means
(e.g., temperature shift or chemical induction), and cells are cultured for an
additional
period. Cells are typically harvested by centrifugation and disrupted by
physical or
chemical means. The resulting crude extract is retained for further
purification. Microbial
cells employed in expression of proteins can be disrupted by any convenient
method,
including freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing
agents.
[067] Various mammalian cell culture systems also can be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7
lines of monkey kidney fibroblasts (Gluzman, Cell 23:175, 1981 ), and other
cell lines
capable of expressing a compatible vector, for example, C127, 3T3, CHO, HeLa,
and
HBK cell lines.
[065] Polypeptides of the present invention may be recovered and purified from
recombinant cell cultures by methods well known in the art, including ammonium
sulfate
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or ethanol precipitation, acid extraction, anion or cation exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxyapatite chromatography, and lectin chromatography. High
performance liquid chromatography (HPLC) can be employed as a final
purification step.
[069] Polypeptides of the invention can be conveniently isolated. A purified
polypeptide
is at least about 70% pure, that is, the isolated polypeptide is substantially
free of cellular
material and has less than about 30% (by dry weight) of non-polypeptide
material.
Preferably, the preparations are 85% through 99% (i.e., 85, 87, 89, 91, 93,
95, 96, 97, 98,
and 99%) pure. Purity of the preparations can be assessed by any means known
in the
art, such as SDS-polyacrylamide gel electrophoresis, mass spectroscopy, and
liquid
chromatography.
[070] Depending upon the host employed in a recombinant production procedure,
polypeptides of the invention may be glycosylated with mammalian or other
eukaryotic
carbohydrates or may be non-glycosylated. Polypeptides of the invention may
also
include an initial methionine amino acid residue.
[071] Alternatively, polypeptides of the invention can be produced using
chemical
methods to synthesize its amino acid sequence, such as by direct peptide
synthesis using
solid-phase techniques (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154,
1963;
Roberge, et al., Science 269:202-204, 1995). Polypeptide synthesis can be
performed
using manual techniques or by automation. Automated synthesis can be achieved,
for
example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer).
Optionally, fragments of a polypeptide can be separately synthesized and
combined
using chemical methods to produce a full-length molecule.
[072] A newly synthesized polypeptide can be substantially purified by
preparative high
performance liquid chromatography (see, e.g., Creighton, Proteins: Structures
And
Molecular Principles, WH Freeman and Co., New York, N.Y., 1983). The
composition of
a synthetic polypeptide of the present invention can be confirmed by amino
acid analysis
or sequencing by, for example, the Edman degradation procedure (see,
Creighton,
supra). Additionally, any portion of the amino acid sequence of the
polypeptide can be
altered during direct synthesis andlor combined using chemical methods with
sequences
from other proteins to produce a variant polypeptide or a fusion polypeptide.
[073] Modified GLP-1 Receptor Aaonists and Methods of Production
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The modified GLP-1 receptor agonists of the present invention comprise a GLP-1
receptor agonist, or a fragment, derivative, variant or analog thereof, which
is linked to a
polyethylene glycol (PEG) polymer having a molecular weight of greater than 30
kD.
[074] Suitable PEG polymers typically are commercially available or may be
made by
techniques well known to those skilled in the art. The PEG polymer has a
molecular
weight of greater than 30 kD, preferably a molecular weight of greater than 30
kD, more
preferably greater than 40 kD, and still more preferably having a branched
structure, such
as for example, a 43 kD branched PEG-peptide (Shearwater 2001 catalog #
2D3XOT01,
mPEG2-MAL).
[075] The attachment of a PEG on an intact peptide can be accomplished by
attaching
the PEG on the opposite side of the peptide surface that interacts with the
receptor.
Preferably, the attachment of PEG will occur on the GLP-1 agonist, numbered in
accordance with GLP-1 (7-37), at positions 22-28 and 30-31, as well as at
positions past
the C terminus; namely positions 32-37. More preferably, attachment of the PEG
will
occur on the GLP-1 agonist, numbered in accordance with GLP-1 (7-37), at
positions 24,
28, 30 and 31, as well as at positions past the C terminus; namely at
positions 32, 34, 36
and 37. Still more preferably, attachment of the PEG will occur on the GLP-1
agonist,
numbered in accordance with GLP-1 (7-37), at the C terminus; namely position
31.
[076] There are several strategies for coupling PEG to peptides (see, e.g.,
Veronese,
Biomaterials 22:405-417, 2001 ), all of which are incorporated herein by
reference in their
entirety. Those skilled in the art, therefore, will be able to utilize such
well-known
techniques for linking the PEG polymer to the GLP-1 receptor agonists
described herein.
[077] Briefly, cysteine PEGylation is one method for site-specific PEGylation,
and it is
often utilized if a peptide has few or no free cysteines. In the case of the
native GLP-1 (7-
37), for example, there are no cysteine residues. Accordingly, PEGylation of
native GLP-
1 (7-37) or any GLP-1 agonist having no cysteine residues can be accomplished
by
introducing a unique cysteine mutation at one of the specific positions on the
native GLP-
1 (7-37) or the GLP-1 agonist identified above and then reacting the resulting
derivative
with a cysteine-specific PEGylation reagent, such as PEG-maleimide.
[078] However, it may be necessary to mutate the GLP-1 agonist of this
invention in
order to allow for site-specific PEGylation. For example, if a GLP-1 agonist
contains
cysteine residues, these will need to be substituted with a conservative amino
acids in
order to ensure site specific PEGylation. In addition, rigid linkers,
including but not limited
to, "GGS" (SEQ ID NO: 32), "GGSGGS" (SEQ ID NO: 33), and "PPPS" (SEQ ID NO:
34),
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may be added to the C-terminus of the GLP-1 agonist, but before the site of
PEG
attachment (i.e., a unique cysteine residue).
[079] Examples of the modified GLP-1 receptor agonists of this invention
include, but are
not limited to, the polypeptides selected from the group consisting of SEQ ID
NOs: 13-14
and 16-31. The most preferred modified GLP-1 receptor agonist of this
invention is SEQ
ID NO: 26.
[080] The polypeptides of the present invention may be employed in treatment
diabetes,
including both type 1 and type 2 diabetes (non-insulin dependent diabetes
mellitus).
Such treatment may also delay the onset of diabetes and diabetic
complications. The
polypeptides may be used to prevent subjects with impaired glucose tolerance
from
proceeding to develop type 2 diabetes. Other diseases and conditions that may
be
treated or prevented using compounds of the invention in methods of the
invention
include: Maturity-Onset Diabetes of the Young (MODY) (Herman, et al., Diabetes
43:40,
1994); Latent Autoimmune Diabetes Adult (LADA) (Zimmet, et al., Diabetes Med.
11:299,
1994); impaired glucose tolerance (IGT) (Expert Committee on Classification of
Diabetes
Mellitus, Diabetes Care 22 (Supp. 1):SS, 1999); impaired fasting glucose (IFG)
(Charles,
et al., Diabetes 40:796, 1991 ); gestational diabetes (Metzger, Diabetes,
40:197, 1991 );
and metabolic syndrome X.
[081] The polypeptides of the present invention may also be efFective in such
disorders
as obesity, and in the treatment of atherosclerotic disease, hyperlipidemia,
hypercholesteremia, low HDL levels, hypertension, cardiovascular disease
(including
atherosclerosis, coronary heart disease, coronary artery disease, and
hypertension),
cerebrovascular disease and peripheral vessel disease.
[082] The compounds of the present invention may also be useful for treating
physiological disorders related to, for example, cell differentiation to
produce lipid
accumulating cells, regulation of insulin sensitivity and blood glucose
levels, which are
involved in, for example, abnormal pancreatic ~i-cell function, insulin
secreting tumors
and/or autoimmune hypoglycemia due to autoantibodies to insulin,
autoantibodies to the
insulin receptor, or autoantibodies that are stimulatory to pancreatic [i-
cells), macrophage
differentiation which leads to the formation of atherosclerotic plaques,
inflammatory
response, carcinogenesis, hyperplasia, adipocyte gene expression, adipocyte
differentiation, reduction in the pancreatic (3-cell mass, insulin secretion,
tissue sensitivity
to insulin, liposarcoma cell growth, polycystic ovarian disease, chronic
anovulation,
hyperandrogenism, progesterone production, steroidogenesis, redox potential
and
oxidative stress in cells, nitric oxide synthase (NOS) production, increased
gamma
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glutamyl transpeptidase, catalase, plasma triglycerides, HDL, and LDL
cholesterol levels,
and the like.
[083] Polypeptides of the invention may also be used in methods of the
invention to treat
secondary causes of diabetes (Expert Committee on Classification of Diabetes
Mellitus,
Diabetes Care 22 (Supp. 1 ):SS, 1999). Such secondary causes include
glucocorticoid
excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes.
Drugs
that may induce diabetes include, but are not limited to, pyriminil, nicotinic
acid,
glucocorticoids, phenytoin, thyroid hormone, (3-adrenergic agents, a-
interferon and drugs
used to treat HIV infection.
[084] The polypeptides of the present invention may be used alone or in
combination
with additional therapies and/or compounds known to those skilled in the art
in the
treatment of diabetes and related disorders. Alternatively, the methods and
compounds
described herein may be used, partially or completely, in combination therapy.
[085] The polypeptides of the invention may also be administered in
combination with
other known therapies for the treatment of diabetes, including PPAR agonists,
sulfonylurea drugs, non-sulfonylurea secretagogues, a-glucosidase inhibitors,
insulin
sensitizers, insulin secretagogues, hepatic glucose output lowering compounds,
insulin
and anti-obesity drugs. Such therapies may be administered prior to,
concurrently with or
following administration of the polypeptides of the invention. Insulin
includes both long
and short acting forms and formulations of insulin. PPAR agonist may include
agonists of
any of the PPAR subunits or combinations thereof. For example, PPAR agonist
may
include agonists of PPAR-a, PPAR-y, PPAR-s or any combination of two or three
of the
subunits of PPAR. PPAR agonists include, for example, rosiglitazone and
pioglitazone.
Sulfonylurea drugs include, for example, glyburide, glimepiride,
chlorpropamide, and
glipizide. a-glucosidase inhibitors that may be useful in treating diabetes
when
administered with a polypeptide of the invention include acarbose, miglitol
and voglibose.
Insulin sensitizers that may be useful in treating diabetes include
thiazolidinediones and
non-thiazolidinediones. Hepatic glucose output lowering compounds that may be
useful
in treating diabetes when administered with a polypeptide of the invention
include
metformin, such as Glucophage and Glucophage XR. Insulin secretagogues that
may be
useful in treating diabetes when administered with a polypeptide of the
invention include
sulfonylurea and non-sulfonylurea drugs: GIP, secretin, nateglinide,
meglitinide,
repaglinide, glibenclamide, glimepiride, chlorpropamide, glipizide. In one
embodiment of
the invention, polypeptides of the invention are used in combination with
insulin
secretagogues to increase the sensitivity of pancreatic (3-cells to the
insulin secretagogue.
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[086] Polypeptides of the invention may also be used in methods of the
invention in
combination with anti-obesity drugs. Anti-obesity drugs include (i-3 agonists,
CB-1
antagonists, appetite suppressants, such as, for example, sibutramine
(Meridia), and
lipase inhibitors, such as, for example, orlistat (Xenical).
[087] Polypeptides of the invention may also be used in methods of the
invention in
combination with drugs commonly used to treat lipid disorders in diabetic
patients. Such
drugs include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic
acid, bile
acid sequestrants, and fibric acid derivatives. Polypeptides of the invention
may also be
used in combination with anti-hypertensive drugs, such as, for example, [3-
blockers and
ACE inhibitors.
[088] Such co-therapies may be administered in any combination of two or more
drugs
(e.g., a compound of the invention in combination with an insulin sensitizer
and an anti-
obesity drug). Such co-therapies may be administered in the form of
pharmaceutical
compositions, as described above.
[089] As used herein, various terms are defined below.
[090] When introducing elements of the present invention or the preferred
embodiments) thereof, the articles "a," "an," "the," and "said" are intended
to mean that
there are one or more of the elements. The terms "comprising," "including,"
and "having"
are intended to be inclusive and mean that there may be additional elements
other than
the listed elements.
[091] The term "subject" as used herein includes mammals (e.g., humans and
animals).
[092] The term "treatment" includes any process, action, application, therapy,
or the like,
wherein a subject, including a human being, is provided medical aid with the
object of
improving the subject's condition, directly or indirectly, or slowing the
progression of a
condition or disorder in the subject.
[093] The term "combination therapy" or "co-therapy" means the administration
of two or
more therapeutic agents to treat a diabetic condition and/or disorder. Such
administration
encompasses co-administration of two or more therapeutic agents in a
substantially
simultaneous manner, such as in a single capsule having a fixed ratio of
active
ingredients or in multiple, separate capsules for each inhibitor agent. In
addition, such
administration encompasses use of each type of therapeutic agent in a
sequential
manner.
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[094] The phrase "therapeutically effective" means the amount of each agent
administered that will achieve the goal of improvement in a diabetic condition
or disorder
severity, while avoiding or minimizing adverse side effects associated with
the given
therapeutic treatment.
[095] The term "pharmaceutically acceptable" means that the subject item is
appropriate
for use in a pharmaceutical product.
[096] Based on well known assays used to determine the efficacy for treatment
of
conditions identified above in mammals, and by comparison of these results
with the
results of known medicaments that are used to treat these conditions, the
effective
dosage of the polypeptides of this invention can readily be determined for
treatment of
each desired indication. The amount of the active ingredient (e.g.,
polypeptides) to be
administered in the treatment of one of these conditions can vary widely
according to
such considerations as the particular compound and dosage unit employed, the
mode of
administration, the period of treatment, the age and sex of the patient
treated, and the
nature and extent of the condition treated.
[097] The total amount of the active ingredient to be administered may
generally range
from about 0.0001 mg/kg to about 200 mg/kg, and preferably from about 0.01
mg/kg to
about 200 mg/kg body weight per day. A unit dosage may contain from about 0.05
mg to
about 1500 mg of active ingredient, and may be administered one or more times
per day.
The daily dosage for administration by injection, including intravenous,
intramuscular,
subcutaneous, and parenteral injections, and use of infusion techniques may be
from
about 0.01 to about 200 mg/kg. The daily rectal dosage regimen may be from
0.01 to
200 mg/kg of total body weight. The transdermal concentration may be that
required to
maintain a daily dose of from 0.01 to 200 mg/kg.
[098] Of course, the specific initial and continuing dosage regimen for each
patient will
vary according to the nature and severity of the condition as determined by
the attending
diagnostician, the activity of the specific polypeptide employed, the age of
the patient, the
diet of the patient, time of administration, route of administration, rate of
excretion of the
drug, drug combinations, and the like. The desired mode of treatment and
number of
doses of a polypeptide of the present invention may be ascertained by those
skilled in the
art using conventional treatment tests.
[099] The polypeptides of this invention may be utilized to achieve the
desired
pharmacological effect by administration to a patient in need thereof in an
appropriately
formulated pharmaceutical composition. A patient, for the purpose of this
invention, is a
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mammal, including a human, in need of treatment for a particular condition or
disease.
Therefore, the present invention includes pharmaceutical compositions which
are
comprised of a pharmaceutically acceptable carrier and a therapeutically
effective amount
of a polypeptide. A pharmaceutically acceptable carrier is any carrier which
is relatively
non-toxic and innocuous to a patient at concentrations consistent with
effective activity of
the active ingredient so that any side effects ascribable to the carrier do
not vitiate the
beneficial effects of the active ingredient. A therapeutically effective
amount of a
polypeptide is that amount which produces a result or exerts an influence on
the
particular condition being treated. The polypeptides described herein may be
administered with a pharmaceutically-acceptable carrier using any effective
conventional
dosage unit forms, including, for example, immediate and timed release
preparations,
orally, parenterally, topically, or the like.
[100] For oral administration, the polypeptides may be formulated into solid
or liquid
preparations such as, for example, capsules, pills, tablets, troches,
lozenges, melts,
powders, solutions, suspensions, or emulsions, and may be prepared according
to
methods known to the art for the manufacture of pharmaceutical compositions.
The solid
unit dosage forms may be a capsule which can be of the ordinary hard- or soft-
shelled
gelatin type containing, for example, surfactants, lubricants, and inert
fillers such as
lactose, sucrose, calcium phosphate, and corn starch.
[101] In another embodiment, the polypeptides of this invention may be
tableted with
conventional tablet bases such as lactose, sucrose, and cornstarch in
combination with
binders such as acacia, cornstarch, or gelatin; disintegrating agents intended
to assist the
break-up and dissolution of the tablet following administration such as potato
starch,
alginic acid, corn starch, and guar gum; lubricants intended to improve the
flow of tablet
granulation and to prevent the adhesion of tablet material to the surfaces of
the tablet
dies and punches, for example, talc, stearic acid, or magnesium, calcium or
zinc stearate;
dyes; coloring agents; and flavoring agents intended to enhance the aesthetic
qualities of
the tablets and make them more acceptable to the patient. Suitable excipients
for use in
oral liquid dosage forms include diluents such as water and alcohols, for-
example,
ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the
addition of a
pharmaceutically acceptable surfactant, suspending agent, or emulsifying
agent. Various
other materials may be present as coatings or to otherwise modify the physical
form of
the dosage unit. For instance tablets, pills or capsules may be coated with
shellac, sugar
or both.
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[102] Dispersible powders and granules are suitable for the preparation of an
aqueous
suspension. They provide the active ingredient in admixture with a dispersing
or wetting
agent, a suspending agent, and one or more preservatives. Suitable dispersing
or
wetting agents and suspending agents are exemplified by those already
mentioned
above. Additional excipients, for example, those sweetening, flavoring and
coloring
agents described above, may also be present.
[103] The pharmaceutical compositions of this invention may also be in the
form of oil-in-
water emulsions. The oily phase may be a vegetable oil such as liquid paraffin
or a
mixture of vegetable oils. Suitable emulsifying agents may be (1 ) naturally
occurring
gums such as gum acacia and gum tragacanth, (2) naturally occurring
phosphatides such
as soy bean and lecithin, (3) esters or partial esters derived from fatty
acids and hexitol
anhydrides, for example, sorbitan monooleate, and (4) condensation products of
said
partial esters with ethylene oxide, for example, polyoxyethylene sorbitan
monooleate.
The emulsions may also contain sweetening and flavoring agents.
[104] Oily suspensions may be formulated by suspending the active ingredient
in a
vegetable oil such as, for example, arachis oil, olive oil, sesame oil, or
coconut oil; or in a
mineral oil such as liquid paraffin. The oily suspensions may contain a
thickening agent
such as, for example, beeswax, hard paraffin, or cetyl alcohol. The
suspensions may
also contain one or more preservatives, for example, ethyl or n-propyl p-
hydroxybenzoate; one or more coloring agents; one or more flavoring agents;
and one or
more sweetening agents such as sucrose or saccharin.
[105] Syrups and elixirs may be formulated with sweetening agents such as, for
example, glycerol, propylene glycol, sorbitol, or sucrose. Such formulations
may also
contain a demulcent, and preservative, flavoring and coloring agents.
[106] The polypeptides of this invention may also be administered
parenterally, that is,
subcutaneously, intravenously, intramuscularly, or interperitoneally, as
injectable dosages
of the compound in a physiologically acceptable diluent with a pharmaceutical
carrier
which may be a sterile liquid or mixture of liquids such as water, saline,
aqueous dextrose
and related sugar solutions; an alcohol such as ethanol, isopropanol, or
hexadecyl
alcohol; glycols such as propylene glycol or polyethylene glycol; glycerol
ketals such as
2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly(ethyleneglycol)
400; an oil; a
fatty acid; a fatty acid ester or glyceride; or an acetylated fatty acid
glyceride with or
without the addition of a pharmaceutically acceptable surfactant such as a
soap or a
detergent, suspending agent such as pectin, carbomers, methycellulose,
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hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent
and other
pharmaceutical adjuvants.
[107] Illustrative of oils which can be used in the parenteral formulations of
this invention
are those of petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil,
soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and
mineral oil.
Suitable fatty acids include oleic acid, stearic acid, and isostearic acid.
Suitable fatty acid
esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps
include fatty
alkali metal, ammonium, and triethanolamine salts and suitable detergents
include
cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl
pyridinium
halides, and alkylamine acetates; anionic detergents, for example, alkyl,
aryl, and olefin
sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and
sulfosuccinates; nonionic
detergents, for example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for
example,
alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium
salts, as well
as mixtures.
[108] The parenteral compositions of this invention may typically contain from
about
0.5% to about 25% by weight of the active ingredient in solution.
Preservatives and
buffers may also be used advantageously. In order to minimize or eliminate
irritation at
the site of injection, such compositions may contain a non-ionic surfactant
having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity
of
surfactant in such formulation ranges from about 5% to about 15% by weight.
The
surfactant can be a single component having the above HLB or can be a mixture
of two or
more components having the desired HLB.
[109] Illustrative of surfactants used in parenteral formulations are the
class of
polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and
the high
molecular weight adducts of ethylene oxide with a hydrophobic base, formed by
the
condensation of propylene oxide with propylene glycol.
[110] The pharmaceutical compositions may be in the form of sterile injectable
aqueous
suspensions. Such suspensions may be formulated according to known methods
using
suitable dispersing or wetting agents and suspending agents such as, for
example,
sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,
sodium
alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting
agents which may be a naturally occurring phosphatide such as lecithin, a
condensation
product of an alkylene oxide with a fatty acid, for example, polyoxyethylene
stearate, a
condensation product of ethylene oxide with a long chain aliphatic alcohol,
for example,
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heptadecaethyleneoxycetanol, a condensation product of ethylene oxide with a
partial
ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol
monooleate,
or a condensation product of an ethylene oxide with a partial ester derived
from a fatty
acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate.
[111] The sterile injectable preparation may also be a sterile injectable
solution or
suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents
and
solvents that may be employed are, for example, water, Ringer's solution, and
isotonic
sodium chloride solution. In addition, sterile fixed oils are conventionally
employed as
solvents or suspending media. For this purpose, any bland, fixed oil may be
employed
including synthetic mono or diglycerides. In addition, fatty acids such as
oleic acid may
be used in the preparation of injectables.
[112] A composition of the invention may also be administered in the form of
suppositories for rectal administration of the drug. These compositions may be
prepared
by mixing the drug (e.g., polypeptide) with a suitable non-irritation
excipient which is solid
at ordinary temperatures but liquid at the rectal temperature and will
therefore melt in the
rectum to release the drug. Such material are, for example, cocoa butter and
polyethylene glycol.
[113] Another formulation employed in the methods of the present invention
employs
transdermal delivery devices ("patches"). Such transdermal patches may be used
to
provide continuous or discontinuous infusion of the compounds of the present
invention in
controlled amounts. The construction and use of transdermal patches for the
delivery of
pharmaceutical agents is well known in the art (see, e.g., U.S. Patent No.
5,023,252,
incorporated herein by reference). Such patches may be constructed for
continuous,
pulsatile, or on demand delivery of pharmaceutical agents.
[114] It may be desirable or necessary to introduce the pharmaceutical
composition to
the patient via a mechanical delivery device. The construction and use of
mechanical
delivery devices for the delivery of pharmaceutical agents is well known in
the art. For
example, direct techniques for administering a drug directly to the brain
usually involve
placement of a drug delivery catheter into the patient's ventricular system to
bypass the
blood-brain barrier. One such implantable delivery system, used for the
transport of
agents to specific anatomical regions of the body, is described in U.S. Patent
No.
5,011,472, incorporated herein by reference.
[115] The compositions of the invention may also contain other conventional
pharmaceutically acceptable compounding ingredients, generally referred to as
carriers or
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diluents, as necessary or desired. Any of the compositions of this invention
may be
preserved by the addition of an antioxidant such as ascorbic acid or by other
suitable
preservatives. Conventional procedures for preparing such compositions in
appropriate
dosage forms can be utilized.
(116] Commonly used pharmaceutical ingredients which may be used as
appropriate to
formulate the composition for its intended route of administration include:
acidifying
agents, for example, but are not limited to, acetic acid, citric acid, fumaric
acid,
hydrochloric acid, nitric acid; and alkalinizing agents such as, but are not
limited to,
ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine,
potassium
hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine,
trolamine.
(117] Other pharmaceutical ingredients include, for example, but are not
limited to,
adsorbents (e.g., powdered cellulose and activated charcoal); aerosol
propellants (e.g.,
carbon dioxide, CCI2F~, F~CIC-CCIF2 and CCIF3); air displacement agents (e.g.,
nitrogen
and argon); antifungal preservatives (e.g., benzoic acid, butylparaben,
ethylparaben,
methylparaben, propylparaben, sodium benzoate); antimicrobial preservatives
(e.g.,
benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium
chloride,
chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and
thimerosal);
antioxidants (e.g., ascorbic acid, ascorbyl palmitate, butylated
hydroxyanisole, butylated
hydroxytoluene, hypophosphorus acid, monothioglycerol, propyl gallate, sodium
ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium
metabisulfite);
binding materials (e.g., block polymers, natural and synthetic rubber,
polyacrylates,
polyurethanes, silicones and styrene-butadiene copolymers); buffering agents
(e.g.,
potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium
citrate anhydrous and sodium citrate dihydrate); carrying agents (e.g., acacia
syrup,
aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orange syrup,
syrup, corn oil,
mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection
and
bacteriostatic water for injection); chelating agents (e.g., edetate disodium
and edetic
acid); colorants (e.g., FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6,
FD&C
Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8, caramel and
ferric
oxide red); clarifying agents (e.g., bentonite); emulsifying agents (but are
not limited to,
acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan
monooleate,
polyethylene 50 stearate); encapsulating agents (e.g., gelatin and cellulose
acetate
phthalate); flavorants (e.g., anise oil, cinnamon oil, cocoa, menthol, orange
oil,
peppermint oil and vanillin); humectants (e.g., glycerin, propylene glycol and
sorbitol);
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levigating agents (e.g., mineral oil and glycerin); oils (e.g., arachis oil,
mineral oil, olive oil,
peanut oil, sesame oil and vegetable oil); ointment bases (e.g., lanolin,
hydrophilic
ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum,
white
ointment, yellow ointment, and rose water ointment); penetration enhancers
(transdermal
delivery) (e.g., monohydroxy or polyhydroxy alcohols, saturated or unsaturated
fatty
alcohols, saturated or unsaturated fatty esters, saturated or unsaturated
dicarboxylic
acids, essential oils, phosphatidyl derivatives, cephalin, terpenes, amides,
ethers,
ketones and ureas); plasticizers (e.g., diethyl phthalate and glycerin);
solvents (e.g.,
alcohol, corn oil, cottonseed oil, glycerin, isopropyl alcohol, mineral oil,
oleic acid, peanut
oil, purified water, water for injection, sterile water for injection and
sterile water for
irrigation); stiffening agents (e.g., cetyl alcohol, cetyl esters wax,
microcrystalline wax,
paraffin, stearyl alcohol, white wax and yellow wax); suppository bases (e.g.,
cocoa butter
and polyethylene glycols (mixtures)); surfactants (e.g., benzalkonium
chloride, nonoxynol
10, oxtoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan
monopalmitate);
suspending agents (e.g., agar, bentonite, carbomers, carboxymethylcellulose
sodium,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, kaolin,
methylcellulose, tragacanth and veegum); sweetening e.g., aspartame, dextrose,
glycerin, mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose);
tablet anti-
adherents (e.g., magnesium stearate and talc); tablet binders (e.g., acacia,
alginic acid,
carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin,
liquid
glucose, methylcellulose, povidone and pregelatinized starch); tablet and
capsule diluents
(e.g., dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline
cellulose,
powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium
phosphate, sorbitol and starch); tablet coating agents (e.g., liquid glucose,
hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose,
ethylcellulose, cellulose acetate phthalate and shellac); tablet direct
compression
excipients (e.g., dibasic calcium phosphate); tablet disintegrants (e.g.,
alginic acid,
carboxymethylcellulose calcium, microcrystalline cellulose, polacrillin
potassium, sodium
alginate, sodium starch glycollate and starch); tablet glidants (e.g.,
colloidal silica, corn
starch and talc); tablet lubricants (e.g., calcium stearate, magnesium
stearate, mineral oil,
stearic acid and zinc stearate); tablet/capsule opaquants (e.g., titanium
dioxide); tablet
polishing agents (e.g., carnuba wax and white wax); thickening agents (e.g.,
beeswax,
cetyl alcohol and paraffin); tonicity agents (e.g., dextrose and sodium
chloride); viscosity
increasing agents (e.g., alginic acid, bentonite, carbomers,
carboxymethylcellulose
sodium, methylcellulose, povidone, sodium alginate and tragacanth); and
wetting agents
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(e.g., heptadecaethylene oxycetanol, lecithins, polyethylene sorbitol
monooleate,
polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).
[118] The polypeptides described herein may be administered as the sole
pharmaceutical agent or in combination with one or more other pharmaceutical
agents
where the combination causes no unacceptable adverse effects. For example, the
polypeptides of this invention can be combined with known anti-obesity, or
with known
antidiabetic or other indication agents, and the like, as well as with
admixtures and
combinations thereof.
[119] The polypeptides described herein may also be utilized, in free base
form or in
compositions, in research and diagnostics, or as analytical reference
standards, and the
like. Therefore, the present invention includes compositions which are
comprised of an
inert carrier and an effective amount of a compound identified by the methods
described
herein, or a salt or ester thereof. An inert carrier is any material which
does not interact
with the compound to be carried and which lends support, means of conveyance,
bulk,
traceable material, and the like to the compound to be carried. An effective
amount of
compound is that amount which produces a result or exerts an influence on the
particular
procedure being performed.
[120] Polypeptides are known to undergo hydrolysis, deamidation, oxidation,
racemization and isomerization in aqueous and non-aqueous environment.
Degradation
such as hydrolysis, deamidation or oxidation can readily detected by capillary
electrophoresis. Enzymatic degradation notwithstanding, polypeptides having a
prolonged plasma half-life, or biological resident time, should, at minimum,
be stable in
aqueous solution. It is essential that polypeptide exhibits less than 10%
degradation over
a period of one day at body temperature. It is still more preferable that the
polypeptide
exhibits less than 5% degradation over a period of one day at body
temperature.
Because of the life time treatment in chronic diabetic patient, much
preferably these
therapeutic agents are convenient to administer, furthermore infrequently if
by parenteral
route. Stability (i.e., less than a few percent of degradation) over a period
of weeks at
body temperature will allow less frequent dosing. Stability in the magnitude
of years at
refrigeration temperature will allow the manufacturer to present a liquid
formulation, thus
avoid the inconvenience of reconstitution. Additionally, stability in organic
solvent would
provide polypeptide be formulated into novel dosage forms such as implant.
[121] Formulations suitable for subcutaneous, intravenous, intramuscular, and
the like;
suitable pharmaceutical carriers; and techniques for formulation and
administration may
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be prepared by any of the methods well known in the art (see, e.g.,
Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20t" edition,
2000).
[122] The following examples are presented to illustrate the invention
described herein,
but should not be construed as limiting the scope of the invention in any way.
[123] Capsule Formulation
A capsule formula is prepared from:
Polypeptide of this invention 10 mg
Starch 109 mg
Magnesium stearate 1 mg
The components are blended, passed through an appropriate mesh sieve, and
filled into
hard gelatin capsules.
[124] Tablet Formulation
A tablet is prepared from:
Polypeptide of this invention 25 mg
Cellulose, microcrystaline 200 mg
Colloidal silicon dioxide 10 mg
Stearic acid 5.0 mg
The ingredients are mixed and compressed to form tablets. Appropriate aqueous
and
non-aqueous coatings may be applied to increase palatability, improve elegance
and
stability or delay absorption.
[125] Sterile IV Solution
A mg/mL solution of the desired compound of this invention is made using
sterile,
injectable water, and the pH is adjusted if necessary. The solution is diluted
for
administration with sterile 5% dextrose and is administered as an IV infusion.
[126] Intramuscular suspension
The following intramuscular suspension is prepared:
Polypeptide of this invention 50 pg/mL ,
Sodium carboxymethylcellulose 5 mg/mL
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TWEEN 80 4 mg/mL
Sodium chloride 9 mg/mL
Benzyl alcohol 9 mg/mL
The suspension is administered intramuscularly.
[127] Hard Shell Capsules
A large number of unit capsules are prepared by filling standard two-piece
hard galantine
capsules each with powdered active ingredient, 150 mg of lactose, 50 mg of
cellulose,
and 6 mg of magnesium stearate.
[128] Soft Gelatin Capsules
A mixture of active ingredient in a digestible oil such as soybean oil,
cottonseed oil, or
olive oil is prepared and injected by means of a positive displacement pump
into molten
gelatin to form soft gelatin capsules containing the active ingredient. The
capsules are
washed and dried. The active ingredient can be dissolved in a mixture of
polyethylene
glycol, glycerin and sorbitol to prepare a water miscible medicine mix.
[129] Immediate Release TabIetsICapsules
These are solid oral dosage forms made by conventional and novel processes.
These
units are taken orally without water for immediate dissolution and delivery of
the
medication. The active ingredient is mixed in a liquid containing ingredient
such as sugar,
gelatin, pectin, and sweeteners. These liquids are solidified into solid
tablets or caplets
by freeze drying and solid state extraction techniques. The drug compounds may
be
compressed with viscoelastic and thermoelastic sugars and polymers or
effervescent
components to produce porous matrices intended for immediate release, without
the
need of water.
[130] It should be apparent to one of ordinary skill in the art that changes
and
modifications can be made to this invention without departing from the spirit
or scope of
the invention as it is set forth herein.
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EXAM PLES
[131] In order that this invention may be better understood, the following
examples are
set forth. These examples are for the purpose of illustration only, and are
not to be
construed as limiting the scope of the invention in any manner. All
publications
mentioned herein are incorporated by reference in their entirety.
[132] Example 1. Peptide Synthesis Methodology.
The following general procedure was followed to synthesize some of the
polypeptides of the invention. Peptide synthesis was carried out by the FMOC/t-
Butyl
strategy (Peptide Synthesis Protocols, Volume 35, Michael W. Pennington & Ben
M.
Dunn, 1994) under continuous flow conditions using Rapp-Polymere PEG-
Polystyrene
resins (Rapp-Polymere, Tubingen, Germany). At the completion of synthesis,
peptides
were cleaved from the resin and de-protected using TFA/DTT/H20/Triisopropyl
silane
(88/5/5/2). Peptides were precipitated from the cleavage cocktail using cold
diethyl ether.
The precipitate was washed three times with the cold ether and then dissolved
in 5%
acetic acid prior to lyophilization. Peptide identity was confirmed by
reversed-phase
chromatography on a YMC-Pack ODS-AQ column (YMC, Inc., Wilmington, NC) on a
Waters ALLIANCE~ system (Waters Corporation, Milford, MA) using
water/acetonitrile
with 3% TFA as a gradient from 0% to 100% acetonitrile, and by MALDI mass
spectrometry on a VOYAGER DETM MALDI Mass Spectrometer, (model 5-2386-00,
PerSeptive BioSystems, Framingham, MA). Matrix buffer (50/50 dH20/acetonitrile
with
3% TFA) peptide sample was added to Matrix buffer 1/1. Those peptides not
meeting the
purity criteria of >95% were purified by reversed-phase chromatography on a
Waters
Delta Prep 4000 HPLC system (Waters Corporation, Milford, MA).
[133] Table 1 contains some selected polypeptides made according to the
Peptide
Synthesis protocols discussed above.
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[134] Table 1
Peptide Peptide Sequence SEQ
ID NO:
GLP-1(7-36) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 1
amide
GLP-17-37 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 2
Exendin 4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH23
G27 HSQGTFTSDYAKYLDARRAKEFIAWLVKGR-NH2 4
G51 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRG 5
G1 HSQGTFTSDYSKYLDSRRAQDFVQWLVKGR-NH2 7
G5 HSQGTFTSDYSKYLEGQAAKEFIAWLVKGR-NH2 8
G55 HSQGTFTSDYARYLDARRAKEFIAWLVKGR-NH2 9
G56 HSQGTFTSDYAAYLDARRAKEFIAWLVKGR-NHS 10
[135] Example 2. Peptide PEG lad
PEGylation may be performed by any method known to those skilled in the art.
However, in this instance, PEGylation was performed using two different
methods to
introduce a unique cysteine mutation into the peptide or at the C-terminus of
the peptide,
followed by PEGylating the cysteine via a stable thioether linkage between the
sulfhydryl
of the peptide and maleimide group of the methoxy-PEG-maleimide reagent
(Inhale/Shearwater). It is preferable to introduce a unique cysteine at the C-
terminus of
the peptide.
[136] In the first method, a 2-fold molar excess of mPEG-mal (MW 22 kD, 30 kD,
and
43 kD) reagent was added to 1 mg peptide and dissolved in reaction buffer at
pH 6
(0.1 M Na phosphate/ 0.1 M NaCI/ 0.1 M EDTA). After 0.5 hour at room
temperature, the
reaction was stopped with 2-fold molar excess of DTT to mPEG-mal. The peptide-
PEG-
mal reaction mixture was applied to a cation exchange column to remove
residual PEG
reagents followed by gel filtration column to remove residual free peptide.
The purity,
mass, and number of PEGylated sites were determined by SDS-PAGE and MALDI-TOF
mass spectrometry.
[137] In the second method, a 10-fold molar excess of mPEG-mal (MW ~22 kD or
43 kD)
reagent was added to 50 pM peptide dissolved in reaction buffer at pH 6 (0.1 M
Na
phosphate! 0.1 M NaCI/ 0.1 M EDTA). After 0.5 hour at room temperature, the
reaction
was stopped with 2-fold molar excess of cysteine over mPEG-mal. The crude
peptide-
PEG-mal reaction mixtures were assayed in vitro without further purification.
[138] Table 2 contains some selected polypeptides made according to the
PEGylation
technology discussed herein. Note that the underlined amino acid represents
the location
that the PEG polymer is attached to the peptide.
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[139] Table 2
Peptide Peptide Seauence SEQ ID
NO:
G71 HSQGTFTSDYAKYLDARRA_CEFIAWLVKGRG 11
G72 HSQGTFTSDYAKYLDARRAKCFIAWLVKGRG 12
G73 HSQGTFTSDYAKYLDARRAKEFI_CWLVKGRG 13
G74 HSQGTFTSDYAKYLDARRAKEFIAWLV_CGRG 14
G75 HSQGTFTSDYAKYLDARRAKEFIAWLVK_CRG 15
G76 HSQGTFTSDYAKYLDARRAKEFIAWLVKG_CG 16
G77 HSQGTFTSDYAKYLDARRAKEFIAWLVKGR_C 17
G78 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRG_C 18
G79 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRGGS_C 19
G80 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRGPPP_C 20
G81 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRGGSGGSC 21
G82 HSQGTFTSDYARYLDARRA_KEFIAWLVRGRG 22
G83 HSQGTFTSDYARYLDARRAREFI_KWLVRGRG 23
G84 HSQGTFTSDYARYLDARRAREFIAWLV_KGRG 24
G85 HSQGTFTSDYARYLDARRAREFIAWLVRGRG_K 25
6185 HSQGTFTSDYARYLDARRAREFIKWLVRGRC 26
[140] Example 3. Measurement of Peptide Signaling Through GLP1 Receptor Using
Cyclic AMP Scintillation Proximity~SPA) Assay
For the modified GLP-1 receptor agonists of this invention, "activation" of
the GLP-
1 receptor in a cAMP scintillation proximity assay is induction of a maximum
activity that
is at least 50%, more preferably at least 70%, still more preferably at least
80%, and still
more preferably at least 90% of the maximal activity induced by the native GLP-
1 (7-36)-
amide. The EC50 value for a modified GLP-1 receptor agonist of this invention
is
between 0.1 and 1000 nM. "EC50" is defined herein as the concentration of a
polypeptide of this invention at which 50% of the maximal activity is
achieved.
[141] RINmSF cells were plated in 96-well plates (Costar) at 1.5 x 105
cells/well and
grown at 37°C for 24 hours in RPMI 1640, 5% FBS, antibiotic/antimycotic
(Gibco BRL).
The media was removed and the cells were washed twice with PBS. The cells were
incubated with peptide concentrations ranging from 1 x 10-'~ to 1 x 10-5 M in
HEPES-PBS
containing 1 % BSA and 100 pM IBMX for 15 minutes at 37°C. The
incubation buffer was
removed, and the cells lysed in lysis reagent provided with the cAMP
Scintillation
Proximity Assay (SPA) direct screening assay system (Amersham Pharmacia
Biotech
Inc, Piscataway, NJ). The amount of cAMP (in pmol) present in the lysates was
determined following instructions provided with this kit. The amount of cAMP
(in pmol)
produced at each concentration of peptide was plotted and analyzed by
nonlinear
regression using Prizm software to determine the EC50 for each peptide.
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[142] The results of this assay with controls (identified with an asterisk)
and
representative polypeptides of this invention are shown in the table below.
[143] Table 3
22 kD 43 kDa
PEG PEG
EC50 EC5 0
PeptideunmodifiedPEG fold-shift unmodifiedPEG fold-shift
G71 27.3 346.1 14.3 33.6 358.6 10.7
*
G72 88.0 476.0 5.2 83.6 411.3 4.9
*
G73 40.4 144.5 4.2 36.0 294.7 8.2
G74 36.0 114.7 3.7 53.8 337.4 7.2
G75 48.3 275.8 7.6 53.7 388.4 8.0
*
G76 20.4 106.6 5.5 26.0 383.0 14.8
G77 26.1 49.7 2.0 22.4 236.1 11.0
G78 15.7 69.5 3.3 20.1 91.6 4.9
G79 19.6 60.2 3.0 16.6 66.9 4.0
G80 14.9 53.2 4.1
G81 20.5 77.6 4.2 16.9 49.6 3.0
[144] Example 4. Insulin Secretion from Dispersed Rat Islet Cells
Increase of insulin secretion from dispersed rat islet cells, in this assay,
is an
increase of at least 1.5 fold. The modified GLP-1 agonist of this invention
increases
insulin secretion from dispersed islet cells by at least about 1.5 fold to
about 10 fold (i.e.,
1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold).
[145] Islets of Langerhans were isolated from SD rats (200-250 g) through a
digestion
procedure using collagenase. Dispersed islet cells were prepared through
treatment with
trypsin, seeded into 96 V-bottom plates and pelleted. Cells were cultured
overnight in
media with and without peptides of the invention. Media was aspirated and the
cells were
pre-incubated with Krebs-Ringer-HEPES buffer containing 3 mM glucose for 30
minutes
at 37°C. Pre-incubation buffer was removed and cells were stimulated
with Krebs-
Ringer-HEPES buffer containing the appropriate glucose concentration (e.g., 8
mM), with
and without peptides for an appropriate time at 37°C. In some studies,
an appropriate
concentration of GLP-1 also is included. A portion of supernatant was removed
and its
insulin content was measured by SPA. The results were expressed as fold over
control
(FOC). At a concentration of 50 nM, the modified GLP-1 agonist having an amino
acid
sequence shown in SEQ ID NO: 26 with a 43 kD PEG linked to the C terminus
increased
insulin secretion from dispersed islet cells approximately 3 fold.
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[146] Example 5. Measuring Increases in Plasma Insulin Levels during IVGTT in
Fasted
Wistar Rats
An increase in plasma insulin levels in this assay is an increase of at least
about
2-fold. Preferably, the modified GLP-1 receptor agonist of this invention
increases insulin
secretion in rats as measured by an increase in plasma insulin levels during
in vivo
glucose tolerance testing in fasted Wistar rats by about 2-fold to about 5-
fold, more
preferably by about 2-fold to about 10 fold, and still more preferably by
about 2-fold to
about 20-fold (i.e., 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13,
14, 15, 16, 17, 18,
19, or 20-fold).
[147] Male Wistar rats will be fasted overnight and then anesthetized with
isoflurane gas.
The rats will be given a tail vein injection of 0.4 g/kg of glucose plus
either vehicle (0.9%
saline + 1% albumin) or 1 nmol/kg GLP-1 (positive control) or 1 nmol/kg of the
polypeptides of this invention. The rats will then be eye-bled one minute
later and the
plasma assayed for insulin using an ELISA Kit (Alpco Diagnostics, Windham,
NH).
[148] Example 6. Effect of PEGylated peptides of this invention on
Intraperitoneal
Glucose Tolerance in Mice
A decrease in blood glucose levels as measured by this assay is a decrease of
at least
about 10%. Preferably, the modified GLP-1 receptor agonist of the invention
decreases
blood glucose levels in mice as measured by intraperitoneal glucose tolerance
testing in
rats or mice by about 10% to about 60%, more preferably by about 10% to about
80%,
still more preferably by about 10% to about 100% (i.e., 10, 20, 30, 40, 50,
60, 70, 80, 90,
or 100%).
[149] The in vivo activity of the modified peptides (PEGylated) of the
invention when
administered subcutaneously was examined in mice. Mice fasted overnight were
given a
subcutaneous injection of control or modified peptide (100 pg/kg). Three hours
later,
basal blood glucose was measured, and the rats were given 2 g/kg of glucose
intraperitoneally. Blood glucose was measured again after 30 and 60 minutes.
[150] The representative modified peptides of the invention significantly
reduced blood
glucose levels relative to the vehicle following the IPGTT, with 36%-54%
reduction in the
glucose AUC ("area under curve"). This demonstrates that modified peptides
have
prolonged glucose lowering activity in vivo. In addition to the glucose
lowering activity of
the modified peptides of the invention, it also indicates prolonged peptide
half-life in vivo.
Unmodified GLP-1 has a very short half-life in vivo (< 10 minUTES). The
ability of the
modified peptides of the invention to lower blood glucose 3 hours following
peptide
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administration is a clear indication that the peptide is present in the
circulation at this time
point and hence has prolonged half-life relative to unmodified GLP-1.
[151] Example 7: Measurement of Gastrointestinal Motility in Mice by
Subcutaneous
In'ection
Modified GLP-1 receptor agonists of this invention have a therapeutic index of
at
least 5-fold. Preferably, the modified GLP-1 receptor agonist of this
invention has a
therapeutic index of about 5-fold to about 10-fold, more preferably by about 5-
fold to
about 20 fold, still more preferably by about 5-fold to about 50-fold, even
more preferable
by about 5-fold to 100-fold, and most preferably by about 5-fold to 200-fold.
The
therapeutic index is the minimum concentration of peptide (i.e., agonist)
required to
reduce gut motility by at least 20% divided by minimum concentration required
to reduce
blood glucose AUC by at least 20% (see Example 6).
[152] Gastrointestinal motility in mice was tested using representative
modified GLP-1
agonists of this invention (GLP-1 agonist linked to a 43 kD PEG, GLP-1 agonist
linked to
a 30 kD PEG, GLP-1 agonist peptides linked to 22 kD PEG, and GLP-1 agonist
linked to
a fatty acid). Gastrointestinal motility was measured as follows: Male Balb/c
mice were
either fasted overnight first and then given peptide (3-100 pg/kg) or vehicle
by
subcutaneous injection, or they were given peptide or vehicle first and then
fasted
overnight, depending on the time interval between dosing and the measurement
of
motility. At the appropriate time after dosing, the mice were given a charcoal
meal by oral
gavage, and then euthanized by cervical dislocation five minutes later. The
small
intestine was dissected out and the length of the intestine measured as well
as the length
the charcoal traveled past the pyloric syphincter. The % traveled was
calculated by
dividing the distance the charcoal traveled by the total length of the small
intestine and
multiplying by 100.
[153] In one example, fatty acid-modified -GLP1 (R34-GLP-1(7-37) with IC26
modified by
y-L-glutamoyl(Na-hexadecanoyl)) was given to overnight fasted mice 3 hours
prior to the
charcoal meal. Fatty acid-GLP1 dose-dependently reduced gastrointestinal
motility
(Figure 3A).
(154] In a second example, GLP-1 linked to a 22 kD PEG was given to overnight
fasted
mice 3 hours prior to the charcoal meal. GLP-1 linked to a 22 kD PEG also dose-
dependently reduced gastrointestinal motility (Figure 3B).
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[155] In a third example, GLP-1 linked to a 43 kD PEG was given to overnight
fasted
mice 3 hours prior to the charcoal meal. GLP-1 linked to a 43 kD PEG showed no
significant effect on gastrointestinal motility (Figure 3B).
[156] In a forth example, GLP-1 agonist linked to a 22 kD PEG was given to
overnight
fasted mice 3 hours prior to the charcoal meal. This GLP-1 agonist linked to a
22 kD
PEG also dose-dependently reduced gastrointestinal motility (Figure 4),
[157] In a fifth example, GLP-1 agonist linked to a 30 kD PEG was given to
overnight
fasted mice 3 hours prior to the charcoal meal. This GLP-1 agonist linked to a
30 kD
PEG also dose-dependently reduced gastrointestinal motility (Figure 4).
[158] In a sixth example, the representative modified GLP-1 agonist of this
invention
(GLP-1 agonist linked to a 43 kD PEG) was given to overnight fasted mice 3
hours prior
to the charcoal meal. The representative modified GLP-1 agonist of this
invention
showed no significant effect on gastrointestinal motility (Figure 4).
[159] Example 8: Measurement of Gastrointestinal Motilit rLin Mice by ICV
Infection
Stainless steel guide cannulas that were aimed at the brain third ventricle
were
implanted to male Wistar rats (275-350 g) anesthetized with isoflurane
anesthesia. The
single 21G cannula was aimed at the brain third ventricle using a stereotaxic
instrument
and the following coordinates: -2.2 mm posterior from bregma and -7.5 mm
ventral to
dura. The cannula was secured to the skull with jeweler's screws and dental
cement.
One week after surgery, cannula placement was tested by infusion of 1 pl
Angiotensin II
at 10 ng/pl concentration. Animals drinking 5 ml or more of water in one-hour
period were
retained for the study. On the day of the gastric motility experiment,
overnight fasted rats
were infused into the third ventricle with vehicle (PBS), 0.5 ~g/rat of GLP-1
(7-36) amide
peptide, or 20 ~g/rat SEQ ID N0:26 modified with a 43 kDa PEG at the C
terminus at
pl volume using an infusion pump (Harvard Apparatus). Peptides or vehicle were
infused during 2 minutes and the injection needle was kept in place an
additional minute
after infusion. Five minutes post infusion, rats received an oral dose of 10%
charcoal,
5% gum arabic, and 1 % carboxymethylcellulose in a volume of 0.8 ml. Five
minutes post
charcoal, the rats were euthanized by CO2 inhalation and decapitated. The
intestines
were dissected out, and the distance the charcoal traveled beyond the pyloric
sphincter
determined. After the experiment, correct placement of the cannula was
verified by
injection of Evans Blue and brain section.
[160] At 20 ~g/rat, SEQ ID N0:26 modified with the 43 kDa PEG produced a
significant
reduction on gastric motility. This effect was comparable to the one produced
by
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0.5 wglrat of GLP-1 (7-36) amide peptide (Figure 5). These data demonstrate
that a GLP-
1 receptor agonist that is linked to a PEG polymer having a molecular weight
of greater
than 30 kD (i.e., the modified GLP-1 receptor agonist of this invention) is
unable to cross
the blood brain barrier, unlike the typical GLP-1 agonist. As a result, the
modified GLP-1
receptor agonist of this invention can prevent a reduction in gastrointestinal
motility,
typically associated with a GLP-1 receptor agonist.
[161] Example 9. Pharmaceutical composition - IV and SC formulations
A sterile IV injectable formulation is made from a derivatized polypetide
(e.g., SEQ
ID NO: 28 linked to a 43 kD PEG) having equivalent of 4 mg polypeptide
content, and
1 liter of sterile saline, using any manufacturing process well known in the
art. Higher
concentration of derivatized polypeptide may be needed for SC formulation.
[162] All publications and patents mentioned in the above specification are
incorporated
herein by reference. Various modifications and variations of the described
compositions
and methods of the invention will be apparent to those skilled in the art
without departing
from the scope and spirit of the invention. Although the invention has been
described in
connection with specific preferred embodiments, it should be understood that
the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed,
various modifications of the above-described modes for carrying out the
invention which
are obvious to those skilled in the field of molecular biology or related
fields are intended
to be within the scope of the following claims. Those skilled in the art will
recognize, or be
able to ascertain using no more than routine experimentation, many equivalents
to the
specific embodiments of the invention described herein. Such equivalents are
intended
to be encompassed by this invention.
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SEQUENCE LISTING
<110> Bayer Pharmaceuticals Corporation
Pan, Clark
Whelan, lames
<120> Modified GLP-1 Receptor Agonists and Their Pharmacological
Methods of Use
<130> MSB-7296
<150> US 60/408,696
<151> 2002-09-16
<150> US 60/439,369
<151> 2003-01-09
<160> 34
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