FIELD OF INVENTION:
The present invention relates to extended GLP-1 compounds. The
invention further relates to a method of stimulating the GLP-1
receptor in a subject in need of such stimulation.
BACKGROUND OF THE INVENTION
A large body of pre-clinical and clinical research data
suggests that glucagon-like pepide-1 (GLP-1) shows great
promise as a treatment for non-insulin dependent diabetes
mellitus (NIDDM) especially when oral agents begin to fail.
GLP-1 induces numerous biological effects such as
stimulating insulin secretion, inhibiting glucagon
secretion, inhibiting gastric emptying, enhancing glucose
utilization, and inducing weight loss. Further, pre-
clinical studies suggest that GLP-1 may also act to prevent
the pancreatic (3 cell deterioration that occurs as the
disease progresses. Perhaps the most salient characteristic
of GLP-1 is its ability to stimulate insulin secretion
without the associated risk of hypoglycemia that is seen
when using insulin therapy or some types of oral therapies
that act by increasing insulin expression.
As NIDDM progresses, it becomes extremely important to
achieve near normal glycemic control and thereby minimize
the complications associated with prolonged hyperglycemia.
GLP-1 would appear to be the drug of choice. However, the
usefulness of therapy involving GLP-1 peptides has been
limited by the fact that GLP-1(1-37) is poorly active, and
the two naturally occurring truncated peptides, GLP-1(7-
37)OH and GLP-1(7-36)NH2, are rapidly cleared in vivo and
have extremely short in vivo half-lives.
Further, GLP-1 peptides currently in development cannot
be given orally and, like insulin, must be injected. Thus,
despite the clear medical advantages associated with therapy
involving GLP-1, the short half-life which results in a drug
that must be injected one or more times a day has impeded
commercial development efforts.
It is known that endogenously produced dipeptidyl-
peptidase IV (DPP-IV) inactivates circulating GLP-1 peptides
by removing the N-terminal histidine and alanine-residues
and is a major reason for the short in vivo half-life.
Thus, recent efforts have focused on the development of GLP-
1 peptides that are resistant to DPP-IV degradation. Some
of these resistant peptides have modifications at the N-
terminus (See U.S. Patent No. 5,705,483), and some are
derivatized GLP-1 peptides wherein large acyl groups that
prevent DPP-IV from accessing the N-.terminus of the peptide
are attached to various amino acids (See WO 98/08871).
The present invention, however, provides a different
approach to the development of biologically active GLP-1
peptides that persist in the serum for extended periods. The
GLP-1 peptides of the present invention are analogs of GLP-
1(7-37) wherein various amino acids are added to the C-
terminus of the analog. These extended GLP-1 peptides not
only have serum half-lives that are much longer than the
native molecules but are particularly suited for oral and
pulmonary administration due to their resistance to various
proteolytic enzymes found in the stomach, intestine, and
lungs. Further, many of these extended GLP-1 peptides are
more potent than the native molecules. This increased
potency coupled with resistance to various proteases
facilitates the use of delivery technology associated with
limited bioavailability. Thus, the present invention makes
possible non-injectable therapy which involves delivering
cost-effective amounts of biologically active GLP-1 peptides
such that therapeutic serum levels are achieved.
It has now been found that a number of GLP-1 peptides
with modifications at various positions coupled with an
extended C-terminus show increased stability compared to
some DPP-IV resistant GLP-1 molecules such as Val8-GLP-1(7-
37) OH. Many of these extended GLP-1 peptides are more patent as
well.
STATEMENT OF THE INVENTION
The present invention relates to an extended GLP-1 peptide
comprising an amino acid sequence of the formula:

wherein the first 31 amino acids of the peptide do not differ from
GLP-1 (7-37) at more than 6 of the corresponding variable positions
and wherein the extended GLP-1 peptide has insulinotropic activity.
DETAILED DESCRIPTION OF THE INVENTION
37)OH. Many of these extended GLP-1 peptides are more
potent as well.
One embodiment of the present invention is a GLP-1
peptide comprising the amino acid sequence of formula 1 (SEQ
ID NO:1)

Formula 1 (SEQ: ID NO: 1)
wherein:
Xaa7 is: L-histidine, D-histidine, desamino-histidine, 2-
amino-histidine, ß-hydroxy-histidine,
homohistidine, a-fluoromethyl -histidine , or a-
methy1-histidine;

Xaa42 is: Pro, Ala, NH2, or is absent;
Xaa43 is: Pro, Ala, NH2, or is absent;
Xaa44 is: Pro, Ala, Arg, Lys, His; NH2, or is absent;
Xaa45 is: Ser, His, Pro, Lys, Arg, Gly, NH2 or is absent;
Xaa46 is: His, Ser, Arg, Lys, Pro, Gly, NH2 or is absent; and
Xaa47 is: His, Ser, Arg, Lys, NH2 or is absent,-
Xaa48 is: Gly, His, NH2 cr is absent;
Xaa49 is: Pro, His, NH2 or is absent; and
Xaa50 is: Ser, His, Ser-NH2, His-NH2 or is absent;
provided that if Xaa42, Xaa43, Xaa44, Xaa45, Xaa46, Xaa47, Xaa48,
or Xaa4S is absent each amino acid downstream is absent and
further provided that the if Xaa36 is Arg and Xaa37 is Gly or
Ser, the GLP-1 peptide does not have the following C-
terminal amino acid extension beginning at Xaa38: Ser-Ser-
Gly-Ala-Pro-Pro-Pro-Ser-NH2.
Another embodiment of the present invention is a GLP-1
peptide comprising the amino acid sequence of formula 2 (SEQ
ID NO:2)
Xaa7-Xaa8-Glu-Gly-Thr-Xaa12-Thr-Ser-Asp-Xaa16-Ser-
Xaa18-Xaa19-Xaa20-Glu-Xaa22-Gln-Ala-Xaa25-Lys-Xaa27-
Phe- Ile-Xaa30-Trp-Leu-Xaa33-Xaa34-Gly-Xaa36-Xaa37-
Xaa38_Xaa39-Xaa40 -Xaa41 -Xaa42_Xaa43_Xaa44-Xaa45-Xaa46_
Xaa47
Formula 2 (SEQ ID NO: 2)
wherein:
Xaa7 is: L-histidine, D-histidine, desamino-histidine, 2-
amino-histidine, ß-hydroxy-histidine,
homohistidine, a-fluoromethyl-histidine, or a-
methyl-histidine;
Xaa8 is: Ala, Gly, Val, Leu, Ile, Ser, or Thr;
Xaa12 is: Phe, Trp, or Tyr;
Xaa16 is: Val, Trp, Ile, Leu, Phe, or Tyr;
Xaa18 is: Ser, Trp, Tyr, Phe, Lys, Ile, Leu, or Val;
Xaa19 is: Tyr, Trp, or Phe;
Xaa20 is: Leu, Phe, Tyr, or Trp;
Xaa22 is: Gly, Glu, Asp, or Lys;
Xaa25 is: Ala, Val, Ile, or Leu;
Xaa27 is: Glu, Ile, or Ala;
Xaa30 is: Ala or Glu
Xaa33 is: Val .or Ile;
Xaa34 is: Lys, Asp, Arg, or Glu;
Xaa36 is: Gly, Pro, or Arg;
Xaa37 is: Gly, Pro, or Ser;
Xaa38 is: Ser, Pro, or His;
Xaa39 is: Ser, Arg, Thr, Trp, or Lys;
Xaa40 is: Ser or Gly;
Xaa4I is: Ala, Asp, Arg, Glu, Lys, or Gly;
Xaa42 is: Pro, Ala, NH2, or is absent;
Xaa43 is: Pro, Ala, NH2, or is absent;
Xaa44 is: Pro, Ala, Arg, Lys, His, NH2, or is absent;
Xaa45 is: Ser, His, Pro, Lys, Arg, NH2 or is absent;
Xaa46 is: His, Ser, Arg, Lys, NH2 or is absent; and
Xaa47 is: His, Ser, Arg, Lys,- NH2 or is absent;
provided that if Xaa42, Xaa43, Xaa44, Xaa45, Xaa46, or Xaa47 is
absent each amino acid downstream is absent and further
provided that if Xaa36 is Arg and Xaa47 is Gly or Ser, the
GLP-1 peptide does not have the following C-terminal amino
acid extension beginning at, Xaa38: Ser-Ser-Gly-Ala-Pro-Pro-
Pro-Ser-NH2.
Another embodiment of the present invention is an
extended GLP-1 peptide comprising the amino acid sequence of
formula 3 (SEQ ID NO:3)
Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Ser-
Tyr-Lys-Glu-Xaa22-Gln-Ala-Xaa2S-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Xaa33-Xaa34-Gly-Xaa36-Xaa37-Xaa38-Xaa39-Xaa4o-
Xaa41-Xaa42-Xaa43 -Xaa44 -Xaa45 - Xaa46 -Xaa47
Formula 3 (SEQ ID NO: 3)
wherein:
Xaa7 is: L-histidine, D-histidine, desamino-histidine, 2-
amino-histidine, p-hydroxy-histidine,
homohistidine, a-fluoromethyl-histidine, or a-
methyl -histidine ;
Xaa8 is: Gly, Val, Leu, He, Ser, or Thr;
Xaa16 is: Val, Trp, Ile, Leu, Phe, or Tyr,-
Xaa22 is: Gly, Glu, Asp, or Lys;
Xaa25 is: Ala, Val, Ile, or Leu;
Xaa33 is: Val or Il;e;
Xaa34 is.: Ly's, Asp, Arg, or Glu;
Xaa36 is: Gly, Pro, or Arg;
Xaa37 is: Gly, Pro, or Ser;
Xaa38 is: Ser, Pro, or His;
Xaa39 is: Ser, Arg, Thr, Trp, or Lys;
Xaa40 is: Ser or Gly;
Xaa41 is: Ala, Asp, Arg, Glu, Lys, or Gly;
Xaa42 is: Pro or Ala,
Xaa43 is: Pro or Ala;
Xaa44 is: Pro, Ala, Arg, Lys, His, NH2, or is absent;
Xaa45 is: Ser, His, Pro, Lys, Arg, NH2 or is absent;
Xaa46 is: His, Ser, Arg, Lys, NH2 or is absent; and
Xaa47 is: His, Ser, Arg, Lys, NH2 or is absent;
provided that if Xaa44, Xaa45, Xaa46, or Xaa47 is absent each
amino acid downstream is absent and further provided that if
Xaa36 is Arg and Xaa37 is Gly or Ser, the GLP-1 peptide does
not have the following G-terminal amino acid extension
beginning at Xaa38: Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH,.
Another embodiment of the present invention is an
extended GLP-1 peptide comprising the amino acid sequence of
formula 4 (SEQ ID NO:4)
Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-
Tyr-Lys-Glu-Xaa22-Gln-Ala-Xaa25-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Xaa33-Lys-Gly-Gly-Pro-Xaa38-Xaa39-Xaa40-Xaa41-
Xaa42-Xaa43-Xaa44- Xaa45-Xaa46-Xaa47
Xaa39 is: Ser, Arg, Thr, Trp, or Lys;
Xaa40 is: Ser or Gly;
Xaa43 is: Ala, Asp, Arg, Glu, Lys; or Gly;
Xaa42 is: Pro, Ala, NH2, or is absent;
Xaa43 is. Pro, Ala, NH2, or is absent;
Xaa44 is: Pro, Ala, Arg, Lys, His, NH2, or is absent;
Xaa45 is: Ser, His, Pro, Lys, Arg, Gly, NH2 or is absent;
Xaa46 is: His, Ser, Arg, Lys, Pro, Gly, NH2 or is absent; and
Xaa47 is: His, Ser, Arg, Lys, NH2 or is absent;
Xaa48 is: Gly, His, NH2 or is absent;.
Xaa49 is: Pro, His, NH2 or is absent; and
Xaa50 is: Ser, His, Ser-NH2, His-NH2 or. is absent,-
wherein said GLP-1 peptide comprises from one to six
further substitutions and provided that if Xaa42, Xaa43,
Xaa44, Xaa45, Xaa46, Xaa47, Xaa48, or Xaa49 is absent each amino
acid downstream is absent
Additional embodiments of formula 1, formula 2, formula
3, formula 4, and formula 5 include GLP-1 peptides that have
valine or glycine at position 8 and glutamic acid at
position 22.
The present invention also encompasses a method of
stimulating the GLP-1 receptor in a subject in need of such
stimulation, said method comprising the step of
administering to the subject, an effective amount of the GLP-
1 peptides described herein. Subjects in need of GLP-1
receptor stimulation include those with non-insulin
dependent diabetes, stress-induced hyperglycemia, and
obesity.
The GLP-1 peptides of the present invention have
various amino acid changes relative to the native GLP-1
molecules and have additional amino acids added to the C-
terminus beginning at position 37.
Native GLP-1(7-37)OH has the amino acid sequence of SEQ
ID NO: 5:
7His-Ala-Glu-10Gly-Thr-Phe-Thr-Ser-15Asp-Val-Ser-Ser-
Tyr-20Leu-Glu-Gly-Gln-Ala-25Ala-Lys-Glu-Phe-Ile-30Ala-
Trp-Leu-Val-Lys-35Gly-Arg-37Gly (SEQ ID NO: 5)
The native molecule is also amidated in vivo such that
the glycine residue at position 37 is replaced with an amide
group. By custom in the art, the amino terminus of GLP-1(7-
37)OH has been assigned residue number 7 and the carboxy-
terminus, number 37. The other amino acids in the
polypeptide are numbered consecutively, as shown in SEQ ID
NO: 4. For example, position 12 is phenylalanine and
position 22 is glycine. The same numbering system is used
for the extended GLP-1 peptides of the present invention.
The GLP-1 peptides encompassed by the present invention
are "extended GLP-1 peptides." Extended GLP-1 peptides have
various amino acid substitutions relative to the native GLP-
1(7-37) or GLP-1(7-36) molecule and have additional amino
acids extending from the C-terminus.
The extended GLP-1 peptides of the present invention
have one or more changes selected from Che following
positions relative to GLP-1(7-37): 7, 8, 12, 16, 18, 19, 20,
22, 25, 27, 30, 33, 34, 36, and 37. In addition, these GLP-
1 peptides have at least 4 amino acids added after amino
acid residue number 37 (Xaa3B through Xaa41) . Preferably, at
least 6 amino acids are added to the C-terminus. Most
preferably between 6 and 10 amino acids are added to the C-
terminus. Even more preferably, between 7 and 9 amino acids
are added to the C-terminus.
The present invention encompasses extended GLP-1
peptides comprising any combination of the amino acids
provided in formula 1 (SEQ ID NO:1), formula 2 (SEQ ID
NO:2), formula 3 (SEQ ID NO:3), and formula 4 (SEQ ID NO:4)
wherein these extended GLP-1 peptides exhibit
"insulinotropic activity." Insulinotropic activity refers
to the ability to stimulate insulin secretion in response to
elevated glucose levels, thereby causing glucose uptake by
cells and decreased plasma glucose levels. Insulinotropic
activity can be assessed by methods known in the art,
including using in vivo experiments and in vitro assays that
measure GLP-1 receptor binding activity or receptor
activation, e.g., assays employing pancreatic isletcells or
insulinoma cells, as described in EP 619,322 to Gelfand, et
al., and U.S. Patent No. 5,120,712, respectively.
Insulinotropic activity is routinely measured in humans by
measuring insulin levels or C-peptide levels.
For the purposes of the present invention an in vitro
GLP-1 receptor signaling assay is used to determine whether
a particular extended GLP-1 peptide will exhibit
insulinotropic activity in vivo. Extended GLP-1 peptides
encompassed by the present invention have an in vitro
potency that is not less than 1/10 the in vitro potency of
the DPP-IV resistant GLP-1 analog known as Val8-GLP-1(7-
37)OH. More preferably, the extended GLP-1 peptides of the
present invention are as potent or more potent than Val8-
GLP-1(7-37)OH.
"In vitro potency" as used herein is the measure of the
ability of a peptide to activate the GLP-1 receptor in a
cell-based assay. In vitro potency is expressed as the
"EC50" which is the effective concentration of compound that
results in 50% activity in a single dose-response
experiment. For the purposes of the present invention, in
vitro potency is determined using a fluorescence assay that
employs HEK-293 Aurora CRE-BLAM cells that stably express
the human GLP-1 receptor. These HEK-293 cells have stably
integrated a DNA vector having a cAMP response element (CRE)
driving expression of the ß-lactamase (BLAM) gene. The
interaction of a GLP-1 agonist with the receptor initiates a
signal that results in activation of the cAMP response
element and subsequent expression of P-lactamase. The ß-
lactamase CCF2/AM substrate that emits fluorescence when it
is cleaved by p-lactamase (Aurora Biosciences Corp.) can
then be added to cells that have been exposed to a specific
amount of GLP-1 agonist to provide a measure of GLP-1
agonist potency. The assay is further described in
Zlokarnik et al. (1998) Science 279:84-88 (See also Example
1). The EC50 values for the compounds listed in example 1
were determined using the BLAM assay described above by
generating a dose response curve using dilutions ranging
from 0.00003 nanomolar to 30 nanomolar. Relative in vitro
potency values are established by running Val8-GLP-1(7-37)OH
as a control and assigning the control a reference value of
1.
Preferably, the extended GLP-1 peptides of the present
invention have the amino acid sequence of GLP-1 (7-37)
modified so that one, two, three, four, five, or six amino
acids differ from the amino, acid in the corresponding
position of GLP-1 (7-37) and in addition have at least 4,
preferably 6, even more preferably between 6 and 10 amino
acids added to the C-terminus.
Preferably, the GLP-1 peptides of the present invention
comprise extended GLP-1 analogs wherein the backbone for
such analogs or fragments contains an amino acid other than
alanine at position 8 (position 8 analogs). The backbone
may also include L-histidine, D-histidine, or modified forms
of histidine such as desamino-histidine, 2-amino-histidine,
P-hydroxy-histidine, homohistidine, a-fluoromethyl-
histidine, or a-methyl-histidine at: position 7. It is
preferable that these position 8 analogs contain one or more
additional changes at positions 12, 16, 18, 19, 20, 22, 25,
27, 30, 33, 34, 36, and 37 compared to the corresponding
amino acid of native GLP-l{7-37). It is more preferable
that these position 8 analogs contain one or more additional
changes at positions 16, 18, 22, 25 and 33 compared to the
corresponding amino acid of native-GLP-1(7-37).
In a preferred embodiment, the amino acid at position
12 of an extended GLP-1 peptide is selected from the group
consisting of tryptophan or tyrosine. It is more preferred
that in addition to the substitution at position 12, the
amino acid at position 8 is substituted with glycine,
valine, leucine, isoleucine, serine,, threonine, or
methionine and more preferably valine or glycine. It is
even more preferred that in addition to the substitutions at
position 12 and 8, the amino acid at position 22 is
substituted with glutamic acid.
In another preferred embodiment, the amino acid at
position 16 of an extended GLP-1 peptide is selected from
the group consisting of tryptophan, isoleucine, leucine,
phenylalanine, or tyrosine. It is preferred that the amino
acid at position 16 is tryptophan. It is more preferred
that in addition to the substitutions at position 16, the
amino acid at position 8 is substituted with glycine,
valine, leucine, isoleucine, serine, threonine, or
methionine and more preferably valine or glycine. It is
even more preferred that in addition to the substitutions at
position 16 and 8, the amino acid at position 22 is
substituted with glutamic acid. It is also preferred that
in addition to the substitutions at positions 16 and 8, the
amino acid at position 33 is substituted with isoluecine. It
is also preferred that in addition to the substitutions at
position 8, 16, and 22, the amino acid at position 36 is
substituted with glycine and the amino acid at position 37
is substituted with proline;
In another preferred embodiment, the amino acid at
position 18 of an extended GLP-1 peptide is selected from
the group consisting of tryptophan, tyrosine, phenylalanine,
lysine, leucine, or isoleucine, preferably tryptophan,
tyrosine, and isoleucine. It is more preferred that in
addition to the substitution at position 18, the amino acid
at position 8 is substituted with glycine, valine, leucine,
isoleucine, serine, threonine, or methionine and more
preferably valine or glycine. It is even more preferred
that in addition to the substitutions at position 18 and 8,
the amino acid at position 22 is substituted with glutamic
acid. It is also preferred, that in addition to the
substitutions at positions .18 and 8, the amino acid at
position 33 is substituted with isoleucine. It is also
preferred that in addition to the substitutions at position
8, 18, and 22, the amino acid at position 36 is substituted
with glycine and the amino acid at position 37 is
substituted with proline.
In another preferred, embodiment, the amino acid at
position 19 of an extended GLP-1 peptide is selected from
the group consisting of tryptophan or phenylalanine,
preferably tryptophan. It is more preferred that in
addition to the substitution at position 19, the amino acid
at position 8 is substituted with glycine, valine, leucine,
isoleucine, serine, threonine, or methionine and more
preferably valine or glycine. It is even more preferred
that in addition to the substitutions at position 19 and 8,
the amino acid at position 22 is substituted with glutamic
acid. It is also preferred that: in addition to the
substitutions at position 8, 19, and 22, the amino acid at
position 36 is substituted with glycine and the amino acid
at position 37 is substituted with proline.
In another preferred embodiment, the amino acid at
position 20 of an extended GLP-1 peptide is selected from
the group consisting of phenylalanine, tyrosine, or
tryptophan, preferably tryptophan. It is more preferred
that in addition to the substitution at position 20, the
amino acid at position 8 is substituted with glycine,
valine, leucine, isoleucine, serine, threonine, or
methionine and more preferably valine or glycine. It is
even more preferred that in addition to the substitutions at
position 20 and 8, the amino acid at position 22 is
substituted with glutamic acid. It is also preferred that
in addition to the substitutions at position 8, 20, and 22,
the amino acid at position 36 is substituted with glycine
and the amino acid at position 37 is substituted with
proline.
In another preferred embodiment, the amino acid at
position 25 of an extended GLP-1 peptide is selected from
the group consisting of valine, isoleucine, and leucine,
preferably valine. It is more preferred that in addition to
the substitution at position 25, the amino acid at position
8 is substituted with glycine, valine, leucine, isoleucine,
serine, threonine, or methionine and more preferably valine
or glycine. It is even more preferred that in addition to
the substitutions at position 25 and 8, the amino acid at
position 22 is substituted with glutamic acid. It is also
preferred that in addition to the substitutions at position
8, 22, and 25, the amino acid at position 36 is substituted
with glycine and the amino acid at position 37 is
substituted with proline.
In another preferred embodiment, the amino acid at
position 27 of an extended GLP-1 peptide is selected from
the group consisting of isoleucine or alanine. It is more
preferred that in addition to the substitution at position
27, the amino acid at position 8 is substituted with
glycine, valine, leucine, isoleucine, serine, threonine, or
methionine and more preferably valine or glycine. It is
even more preferred that in addition to the substitutions at
position 27 and 8, the amino acid at position 22 is
substituted with glutamic acid. It is also preferred that
in addition to the substitutions at position 8, 22, and 27,
the amino acid at position 36 is substituted with glycine
and the amino acid at position 37 is substituted with
proline.
In another preferred embodiment, the amino acid at
position 33 of an extended GLP-1 peptide is isoleucine. It
is more preferred that in addition to the substitution at
position 33, the amino acid at position 8 is substituted
with glycine, valine, leucine, isoleucine, serine,
threonine, or methionine and more preferably valine or
glycine. It is even more preferred that in addition to the
substitutions at position 33 and 8, the amino acid at
position 22 is substituted with glutamic acid. It is also
preferred that in addition to the substitutions at position
8, 22, and 33 the amino acid at position 36 is substituted
with glycine and the amino acid at position 37 is
substituted with proline.
In another preferred embodiment, the amino acid at
position 34 is aspartic acid. It is more preferred that in
addition to the substitution at position 34, the amino acid
at position 8 is substituted with glycine, valine, leucine,
isoleucine, serine, threonine, or methionine and more
preferably valine or glycine. It is even more preferred
that in addition to the' substitutions at position 34 and 8,
the amino acid at position 22 is substituted with glutamic
acid. It is also preferred that in addition to the
substitutions at position 8, 22, and 34 the amino acid at
position 36 is substituted with glycine and the amino acid
at position 37 is substituted with proline.
The C-terminal extension portion fused to the GLP-1
analog backbones discussed above is at least 4 amino acids
in length, preferably between 6 and 10 amino acids in
length. Preferably, the extended GLP-1 peptides of the
present invention have a serine, proline, or histidine at
position 38; a serine, arginine, threonine, tryptophan, or
lysine at position 39; a serine or glycine at position 40;
an alanine, aspartic acid, arginine, glutamic acid, lysine
or glycine at position 41; a proline or alanine at position
42; and a proline or alanine at position 43. Additional
amino acids that may be added include a proline, serine,
alanine, arginine, lysine or histidine at position 44; a
serine,' histidine, proline, lysine or arginine at position
45; a histidine, serine, arginine, or lysine at position 46;
and a histidine, serine, arginine, or lysine at position 47.
Preferably, histidine is the C-terminal amino acid at either
position 42, 43, 44, 45, 46, or 47. Additional amino acids
that may be added to the C-terminus also include those
specified in formula 1 (SEQ ID NO:l).
It is preferred that when Xaa34 is aspartic acid, then
Xaa41 is arginine or lysine. It is also preferred that Xaa39
is serine. It is also preferred that when Xaa4i is aspartic
acid or arginine, then Xaa42, Xaa43, and Xaa44 are all
proline. The C-terminal amino acid may be in the typical
acid form or may be amidated.
A preferred genus of extended GLP-1 peptides comprise
the amino acid sequence of formula 4 (SEQ ID NO:4)
Xaa-, -Xaa8-Glu-Gly-Thr-Phe -Thr-Ser-Asp- Val-Ser-Ser-
Tyr-Lys-Glu-Xaa22-Gln-Ala-Xaa25-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Xaa33-Lys-Gly-Gly-Pro-Xaa38-Xaa39-Xaa40-Xaa41-
Xaa42-Xaa43-Xaa44-Xaa45-Xaa46-Xaa47
Formula 4 (SEQ ID NO:4)
wherein:
Xaa7 is: L-histidine, D-histidine, desamino-histidine, 2- amino-hist idine , ß-hydroxy-histidine ,
homohistidine, a-fluoromethyl-histidine, or a-
methyl-histidine;
Xaa8 is: Gly, Val, Leu, Ile, Ser, or Thr;
Xaa22 is: Gly, Glu, Asp, or Lys;
Xaa25 is: Ala, Val, Ile, or Leu;
Xaa33 is: Val or Ile;
Xaa38 is: Ser, Pro, or His;
Xaa39 is: Ser, Arg, Thr, Trp, or Lys;
Xaa40 is: Ser or Gly;
Xaa41 is: Ala, Asp, Arg, Glu, Lys, or Gly;
Xaa42 is: Pro or Ala;
Xaa43 is: Pro or Ala;
Xaa44 is: Pro, Ala, Arg, Lys, His, NH2, or is absent;
Xaa45 is: Ser, His, Pro, Lys,, Arg, NH2 or is absent;
Xaa46 is:His, Ser, Arg, Lys, NH2 or is absent; and
Xaa47 is: His, Ser, Arg, Lys, NH2 or is absent;
provided that if Xaa44, Xaa45, Xaa46, or Xaa47 is absent each
amino acid downstream is absent.
Preferred extended GLP-1 peptides are peptides of
formula 4 (SEQ ID NO:4) wherein Xaa7 is L-His, Xaa8 is Gly
or Val, Xaa22 is Glu, Xaa25 ;is Val, Xaa33 is lie, Xaa38 is
Ser, Xaa39 is Ser, Xaa4o is Gly, Xaa41 is Ala, Xaa42 is Pro,
Xaa43 is Pro, Xaa44 is Pro, Xaa45 is Ser and Xaa46 and Xaa47
are absent and amidated forms of thereof. Preferred
extended GLP-1 peptides also include peptides of formula 4
(SEQ ID NO: 4) wherein Xaa7 is L-His, Xaa8 is Val, Xaa22 is
Glu, Xaa25 is Ala, Xaa33 is Il Xaa38 is Ser, Xaa39 is Ser,
Xaa40 is Gly, Xaa41 is Ala, Xaa42 is. Pro, Xaa43 is Pro, Xaa44
is Pro, Xaa45 is Ser and Xaa46and Xaa47 are absent and
amidated forms thereof. Other preferred extended GLP-1
peptides include peptides of formula 4 (SEQ ID NO:4) wherein
Xaa7 is L-His, Xaa8 is Val, Xaa22 is Gly, Xaa25 is Ala, Xaa33
is Il Xaa3B is Ser, Xaa39 is. Ser, Xaa40 is Gly, Xaa41 is
Ala, Xaa42 is Pro, Xaa43 is Pro, Xaa44 is Pro, Xaa45 is Ser,
and Xaa46 and Xaa47 are absent and amidated forms thereof.
The present invention encompasses the discovery that
specific amino acids added to the G-terminus of a GLP-1
peptide provide specific structural features that protect
the peptide from degradation by various proteases yet do not
negatively impact the biological activity of the peptide.
Further, many of the extended peptides disclosed herein are
more potent than DPP-IV resistant GLP-l analogs such as
Val8-GLP-1(7-37)OH.
Example 1 provides in vitro potency data for a
representative number of extended GLP-l peptides. The in
vitro potency of the tested extended GLP-l peptides ranged
from about the same as Val8-GLP-1(7-37)OH to greater than 7-
,fold more potent than Val8-GLP-1(7-37)OH. Further, example
5 illustrates that extended GLP-l peptides are also more
potent in vivo.
Example 2 provides a measure of protease insensitivity
for a representative number of extended GLP-l analogs. The
relative proteolytic stability was determined by exposing
extended GLP-l peptides and Val8-GLP-1(7-37)OH to a-
chymotrypsin and then plotting the progress of the enzymatic
reaction as described in Example 2. The extended GLP-l
peptides tested ranged from as stable as Val8-GLP-1(7-37)OH
to 5-fold more stable than Val8-GLP-l(7-37)OH.
The; extended GLP-l peptides of the present invention
also have an increased half-life in vivo as indicated in
example 4. The in vivo half-life of these extended peptides
is generally longer than the half-life of DPP-IV protected
GLP-l analogs such as Val8-GLP-1(7-37)OH.
The extended GLP-l peptides of the present invention
are suited for oral administration, nasal administration,
pulmonary inhalation or parenteral administration.
Parenteral administration can include, for example,
systemic administration, such as by intramuscular,
intravenous, subcutaneous, or intraperitoneal injection.
The GLP-l compounds can be administered to the subject in
conjunction with an acceptable pharmaceutical carrier,
diluent or excipient as part of a pharmaceutical
composition for treating various diseases and conditions
discussed herein. The pharmaceutical composition can be
a solution or a suspension'. Suitable pharmaceutical
carriers may contain inert ingredients which do not
iinteract with the peptide or peptide derivative.
Standard pharmaceutical formulation techniques may be
employed such as those described in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton,
PA. Suitable pharmaceutical carriers for parenteral
administration include, for example, sterile water,
physiological saline, bacteriostatic saline (saline
containing about 0.9% mg/ml benzyl alcohol), phosphate-
buffered saline, Hank's solution, Ringer's-lactate and
the like. Some examples of suitable excipients include
lactose, dextrose, sucrose, trehalose, sorbitol, and
mannitol.
The GLP-1 compounds may be formulated for
administration such that blood plasma levels are
maintained in the efficacious range for extended time
periods. For example, depot formulations wherein a
bioadsorbable polymer is used to provide sustained
release over time are also suitable for use in the
present invention.
The main barrier to effective oral peptide drug
delivery is poor bioavailability due to degradation of
peptides by acids and enzymes, poor absorption through
epithelial membranes, and transition of peptides to an
insoluble form after exposure to the acidic pH
environment in the digestive tract. This reduced
bioavailability necessitates the use of GLP-1 compounds
with increased potency, increased stability, or both.
Oral delivery systems for peptides such as those
encompassed by the present invention are known in the
art. For example, GLP-1 compounds can be encapsulated
using microspheres or other carriers and then delivered
orally.
The extended GLP-1 peptides described herein can be
used to treat subjects with a wide variety of diseases and
conditions. The extended GLP-1 peptides encompassed by the
present invention exert their biological effects by acting
at a receptor referred to as the "GLP-1 receptor" (see
Dillon, J.S. et al. (1993), Endocrinology, 133: 1907-1910).
Subjects with diseases and/or conditions that respond
favorably to GLP-1 receptor stimulation or to the
administration of extended GLP-1 peptides can therefore be
treated. These subjects are said to "be in need of
treatment with extended GLP-1 peptides" or "in need of GLP-1
receptor stimulation".
Included are subjects with non-insulin dependent
diabetes, insulin dependent diabetes, stress-induced
hyperglycemia, stroke (see WO 00/16797 by Efendic),
myocardial infarction (see WO 98/08531 by Efendic),
catabolic changes after surgery (see U.S. Patent No.
6,006,75:3 to Efendic), functional dyspepsia and irritable
bowel syndrome (see WO 99/64060 by Efendic). Also included
are subjects requiring prophylactic treatment with a GLP-1
peptide, e.g., subjects at risk for developing non-insulin
dependent diabetes (see WO 00/07617) . Additional subjects
include those with impaired glucose tolerance or impaired
fasting glucose, subjects with a partial pancreatectomy,
subjects having one or more parents with non-insulin
dependent diabetes, subjects who have had gestational
diabetes and subjects who have had acute or chronic
pancreatitis and are at risk for developing non-insulin
dependent diabetes.
The extended GLP-1 peptides of the present invention
are also useful in treating subjects who are overweight.
Particularly suited are those subjects whose body weight is
about 25% above normal body weight for the subject's height
and body build. Thus, the extended GLP-1 peptides can also
be used to treat obesity (see WO 98/19698 by Efendic).
The extended GLP-1 peptides of the present invention
can be used to normalize blood glucose levels, prevent
pancreatic P-cell deterioration, induce P-cell
proliferation, stimulate insulin gene transcription, up-
regulate IDX-l/PDX-1 or other growth factors, improve P-cell
function, activate dormant P-cells, differentiate cells into
P-cells, stimulate P-cell replication, inhibit P-cell
apoptosis, regulate body weight, and induce weight loss.
An "effective amount" of an extended GLP-1 peptide is
the quantity which results in a desired therapeutic and/or
prophylactic effect without causing unacceptable side-
effects when administered to a subject in need of GLP-1
receptor stimulation. A "desired therapeutic effect"
includes one or more of the following: 1) an amelioration of
the symptom(s) associated with the disease or condition; 2)
ia delay in the onset of symptoms associated with the disease
or condition; 3) increased longevity compared with the
absence of the treatment; and 4) greater quality of life
compared with the absence of the treatment. For example, an
"effective amount" of an extended GLP-1 peptide for the
treatment of type 2 diabetes is the quantity that would
result in greater control of blood glucose concentration
than in the absence of treatment, thereby resulting in a
delay in the onset of diabetic complications such as
retinopathy, neuropathy or kidney disease. An "effective
amount" of an extended GLP-1 peptide for the prevention of
diabetes is the quantity that would delay, compared with the
absence of treatment, the onset of elevated blood glucose
levels that require treatment with drugs such as
sulfonylureas, thiazolidinediones, insulin and/or
bisguanidines.
A typical dose range tor the extended GLP-1 peptides of
the present invention, will range from about 1 ug to about
100 mg per day. Preferably, the dose range is about 5 µg to
about 1 mg per day. Even more preferably the dose is about
10 ug to about 100 ug per day.
A "subject" is a mammal, preferably a human, but
can also be an animal, e.g., companion animals (e.g.,
dogs, cats, and the like), farm animals (e.g., cows,
sheep, pigs, horses, and the, like) and laboratory
animals (e.g., rats, mice, guinea pigs, and the like).
The extended GLP-1 peptides of the present invention
pan be prepared using recombinant DNA technology or by using
[standard methods of solid-phase peptide synthesis
techniques. Peptide synthesizers are commercially available
from, for example, Applied Biosystems in Foster City CA.
Reagents for solid phase synthesis are commercially
available, for example, from Midwest Biotech (Fishers, IN).
Solid phase peptide synthesizers can be used according to
manufacturers instructions for blocking interfering groups,
protecting the amino acid to be reacted, coupling,
decoupling, and capping of unreacted amino acids.
Typically, an a-N-carbamoyl protected amino acid and
the N-terminal amino acid on the growing peptide chain on a
resin is coupled at room temperature in an inert solvent
such as dimethylformamide, N-methylpyrrolidone or methylene
chloride in the presence of coupling agents such as
dicyclohexylcarbodiimide and 1-hydroxybenzotriazole and a
base such as diisopropylethylamine. The a-N-carbamoyl
protecting group is removed from the resulting peptide resin
using a reagent such as trifluoroacetic acid or piperidine,
and the coupling reaction repeated with the next desired N-
protected amino acid to be added to the peptide chain.
Suitable amine protecting groups are well known in the art
and are described, for example, in Green and Wuts,
"Protecting Groups in Organic Synthesis", John Wiley and
Sons, 1991, the entire teachings of which are incorporated
by reference. Examples include t-butyloxycarbonyl (tBoc) and
fluorenylmethoxycarbonyl (Fmoc).
After completion of synthesis, peptides are cleaved
from.the solid-phase support with simultaneous side-chain
deprotection using standard hydrogen fluoride or
trifluoroacetic acid cleavage protocols. Crude peptides are
then further purified using Reversed-Phase Chromatography on
Vydac C18 columns employing linear water-acetonitrile
gradients with all solvents containing 0.1% trifluoroacetic
acid (TFA) . To remove acetpnitrile, peptides are
lyophilized from a solution containing 0.1 % TFA,
acetonitrile and water. Purity can be verified by
analytical reversed phase chromatography. Identity of
peptides can be verified by mass spectrometry. Peptides can
be solubilized in aqueous buffers at neutral pH.
EXAMPLES
Example 1
In vitro potency:
HEK-293 Aurora CRE-BLAM eel 2 s expressing the human GLP-
1 receptor are seeded at 20,000 to .40,000 cells/well/100 µl
into a 96 well black clear bottom plate. The day after
seeding, the medium is replaced with plasma free medium. On
the third day after seeding, 20 µl of plasma free medium
containing different concentrations of GLP-1 agonist is
added to each well to generate a dose response curve.
Generally, fourteen dilutions containing from 3 nanomolar to
30 nanomolar GLP-1 compound were used to generate a dose
response curve from which EC50 values could be determined.
Example 2
Proteolytic stability:
The relative susceptibility of various extended GLP-1
peptides to a-chytnotrypsin was assessed in a reaction
mixture with the control peptide Val8-GLP-1 (7-37) OH. A 10
mM phosphate/citrate solution, pH 7.4, was prepared
containing GLP-1 peptides at a concentration of 100 p.M. A
10 µl aliquot of this solution was then incubated at 4°C in
a 200 ul 10 mM phosphate/citrate solution, pH 7.4,
containing 10 mM CaCl2. Alpha-Chymotrypsin (SIGMA, C-3142
lot 89F8155) was then added to a final concentration of 250
ng/ml . A 20 µl aliquot was injected onto an analytical
Zorbax 300SB-C8 (4.6 mm i.d. x 50 mm) column at a 1 ml/min
flowrate in 10% acetonitrile/0.075% TFA before addition of
the enzyme, as well as 20/40, 60, 80, and 100 minutes
following addition of the enzyme.'. Peaks were separated with
a gradient of 10 to 90% acetonitrile/0.075% TFA over 15 min.
The progress of the enzymatic reaction was followed by
plotting loss of peak area of the starting material over
time'. The rate of proteolytic degradation was calculated
from the initial rate of cleavage (timepoint 0 and 20 min)
and directly compared to the rate of cleavage of the control
peptide Val8-GLP-1 (7-37) OH. The following extended GLP-1
peptides were tested and had stability rates ranging from
about the same as to greater than 5-fold more stable than
Example. 3
Physical stability:
Extended GLP-1 peptides were analyzed with respect to
their potential to aggregate in solution. In general,
peptides in solution were stirred at elevated temperature in
a suitable buffer while recording turbidity at 350 nm as a
function of time. Time to the onset of aggregation was
measured to quantify the potential of a given GLP molecule
to aggregate under these stressed conditions.
A GLP-1 peptide was first dissolved under alkaline
conditions (pH 10.5) for 30 minutes to dissolve any pre-
aggregated material. The solution was then adjusted to pH
7.4 and filtered. Specifically, 4 mg of a lyophilized GLP-1
compound was dissolved in 3 ml of 10 mM phosphate/10 mM
citrate. The pH was adjusted to 10.0-10.5 and held for 30
minutes. The solution was adjusted with HCl to pH 7.4 and
filtered through a suitable filter, for example a Millex GV
syringe filter (Millipore Corporation, Bedford, MA). This
solution was then diluted to a final sample containing 0.3
mg/mL protein in 10 mM citrate, 10 mM phosphate, 150 mM
NaCl, and adjusted to pH 7.4 to 7.5. The sample was
incubated at 37°c in a quartz cuvette. Every five minutes
the turbidity of the solution was measured at 350 nm on an
AVIV Model 14DS UV-VIS spectrophotometer (Lakewood, NJ).
For 30 seconds prior to and during the measurement the
solution was stirred using a magnetic stir bar from Starna
Cells, Inc. (Atascadero, CA). An increase in OD at 350 nm
indicates aggregation of the. GLP-peptide. The time to
aggregation was approximated by the intersection of linear
fits to the pre-growth and growth phase according to the
method of Drake (Arvinte T, Cudd A, and Drake AF. (1993; J.
Biol. Chem. 268, 6415-6422).
The cuvette was cleaned between experiments, with a
caustic soap solution (e.g., Contrad-70). The following
extended GLP-1 peptides were tested and were stable in
solution for at least 55 hours compared to Val8-GLP-1(7-
37)OH which was stable for about 1 hour:
Example 5
In vivo activity of extended :GLP-1 peptides:
Several different extended and non-extended GLP-1
peptides were tested for activity in a hyperglycemic clamp
study in dogs. Glucose was infused for 200 minutes to
maintain constant levels. For the first 80 minutes dogs
were infused intravenously with vehicle to establish a
baseline insulin concentration. For the next 60 minutes,
GLP-1 peptides were administered at a rate of 1 pmol/kg/min.
For the final 60 minutes the infusion rate, of each GLP-1
compound was increased to 3 pmol/kg/min. Blood samples were
taken periodically for the determination of insulin and GLP-
1 peptide concentrations. Insulin change values were
calculated as the difference between the value at time t and
the average value during the last 20 minutes of the control
period (60-80) minutes and are presented in Table 3. Areas
under the insulin change curves were calculated using the
trapezoidal rule over the last 30 minutes of each infusion
period. GLP-1 peptide concentrations are presented in Table
4. Values listed are the means ± standard error of the mean
(SEM).
WE CLAIM:
1. An extended GLP-1 peptide comprising an amino acid sequence of the formula:
Xaa7-Xaa8-Glu-Gly-Thr-Xaal2-Thr-Scr-Asp-Xaal6-Ser-Xaal8-Xaal9-Xaa20-Glu-
Xaa22-GIn-Ala-Xaa25-Lys-Xaa27-Phe-Ile-Xaa30-Trp-Leu-Xaa33-Xaa34-Gly-
Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46-
Xaa47-Xaa48-Xaa49-Xaa50 Formula 1 (SEQ ID NO: 1)
wherein:
Xaa7 is: L-histidine;
Xaa8 is: Gly. or Val;
Xaal2 is:-Phe;
Xaa16 is: Val. Trp, Ile, Leu, Phe, or Tyr;
Xaa18 is: Ser;
Xaa19 is:Tyr;
Xaa20 is: Leu, Phe, Tyr, or Trp;
Xaa22 is: Glu;
Xaa25 is: Ala, Val, Ile, or Leu;
Xaa27 is:Glu. Ile, or Ala;
Xaa30 is: Ala. or Glu
Xaa33 is: Val. or Ile:
Xaa34 is: Lys, Asp, Arg, or Glu;
Xaa36 is: Gly. Pro. or Arg;
Xaa37 is: Gly. Pro, or Ser;
Xaa38 is: Ser. Pro, or His;
Xaa39 is: Ser, Arg, Thr, Trp, or Lys;
Xaa40 is: Ser, or Gly;
Xaa41 is: Ala, Asp, Arg, Glu, Lys, or Gly;
Xaa42 is: Pro. or Ala;
Xaa43 is: Pro, or Ala;
Xaa44 is: Pro, Ala, Arg, Lys, or His;
Xaa45 is: Ser, His, Pro, Lys, Arg, or Gly;
Xaa46 is: His. Ser, Arg, Lys, Pro, or Gly;
Xaa47 is: His, Ser, Arg, Lys, NH2 or is absent;
Xaa48 is: Gly. His, NH2 or is absent;
Xaa49 is: Pro. His. NH2 or is absent; and
Xaa50 is: Ser, Mis. Ser-NH2, His-NH2 or is absent; and
wherein the first 31 amino acids of the peptide do not differ from GLP-1 (7-37) at
more than 6 of the corresponding variable positions and wherein the extended GLP-1
peptide has insulinotropic activity.
2. The GLP-1 peptide as claimed in claim 1, wherein the first 31 amino acids of the
peptide do not differ from GLP-1 (7-37) at more than 5 of the corresponding variable
positions.
3. The GLP-1 peptide as claimed in claim 3. wherein the first 31 amino acids of the
peptide do not differ from GLP-1 (7-37) at more than 4 of the corresponding variable
positions.
4. The GLP-1 peptide as claimed in claim 4. wherein the first 31 amino acids of the
peptide do not differ from GLP-1 (7-37) at more than 3 of the corresponding variable
positions.
5. The GLP-1 peptide as claimed in claim 1, wherein Xaa1 6 is Trp.
6. The GLP-1 peptide as claimed in claim 1, wherein Xaa25 is Val.
7. The GLP-1 peptide as claimed in claim 1, wherein Xaa33 is lie.
8. The GLP-1 peptide as claimed in claim 1, wherein Xaa34 is Asp.
9. The GLP-1 peptide as claimed in claim 13, wherein Xaa41 is Arg.
10. The GLP-1 peptide as claimed in claim 1, wherein Xaa36 is Gly, and Xaa37 is Pro.
11. The GLP-1 peptide as claimed in claim 1, wherein Xaa18 is Trp.
12. The GLP-1 peptide as claimed in claim 1, wherein Xaa20 is Trp.
13. The GLP-1 peptide as claimed in claim 1, wherein the C-terminal amino acid is
amidated.
14. The GLP-1 peptide as claimed in claim. 1. wherein the C-terminal amino acid is His.
15. An extended GLP-1 substantially such as herein described and illustrated in the
forgoing examples.

The invention encompasses GLP-1 peptides with modifications at various positions coupled
with an extended C-tcrminus that provides increased stability.