GIP AND GLP-1 CO-AGONIST COMPOUNDS
The present invention relates to the field of medicine. More particularly, the
present invention relates to dual incretin peptide mimetic compounds that agonize
receptors for both human glucose-dependent insulinotropic polypeptide (GIP) and
glucagon-like peptide- 1 (GLP-1), and may be useful for treating type 2 diabetes mellitus
(T2D).
T2D is the most common form of diabetes accounting for approximately 90% of
all diabetes. T2D is characterized by high blood glucose levels caused by insulinresistance.
The current standard of care for T2D includes diet and exercise along with
available oral and injectable glucose lowering drugs. Nonetheless, many patients with
T2D still remain inadequately controlled. Currently marketed incretin mimetics or
dipeptidyl peptidase IV (DPP-IV) inhibitors only utilize a single established mechanism
of action for glycemic control. A compound for T2D is needed that utilizes a dual
mechanism of action.
GIP is a 42-amino acid gastrointestinal regulatory peptide that plays a
physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic
beta cells in the presence of glucose and protecting pancreatic beta cells. GLP-1 is a 37-
amino acid peptide that stimulates insulin secretion, protects pancreatic beta cells, and
inhibits glucagon secretion, gastric emptying and food intake which leads to weight loss.
GIP and GLP-1 are known as incretins; incretin receptor signaling exerts physiologically
relevant action critical for glucose homeostasis. In normal physiology, GIP and GLP-1
are secreted from the gut following a meal, and these incretins enhance the physiological
response to food including sensation of satiety, insulin secretion, and nutrient disposal.
T2D patients have impaired incretin responses.
Dosing of GLP-1 analogues has been found to be limited by adverse effects, such
as nausea and vomiting, and as a consequence dosing most often cannot reach full
efficacy for glycemic control and weight loss. GIP alone has very modest glucoselowering
ability in type 2 diabetic humans. Both native GIP and GLP-1 are inactivated
rapidly by the ubiquitous protease, DPP IV, and therefore, can only be used for short-term
metabolic control.
Glucagon is a 29-amino acid peptide produced by the pancreas, and when bound
to glucagon receptor, signals the liver to release glucose leading to an increase in blood
glucose. GLP-2, a peptide that like GLP-1 is produced from processing of proglucagon,
is known to be associated with cellular proliferation in the gut. Thus, stimulation of
glucagon and GLP-2 receptors should be minimized during chronic treatment of T2D
patients in order to maximize glucose lowering and reduce potential long term
carcinogenic risks.
Certain GIP analogs have been described as exhibiting both GIP and GLP- 1
activity in WO 2013/164483, WO 2014/192284, and WO 2011/119657.
DPP IV is in the exopeptidase class of proteolytic enzymes. The introduction of
non-natural amino acids in a sequence can increase the proteolytic stability of any given
peptide. While use of non-natural amino acids can help with the stability of peptides
against DPP IV proteolysis and other forms of degradation, it was discovered by
Applicants as part of the present invention that non-natural amino acids can have
unexpected effects on the balance of agonist activity between GIP and GLP-1. Nonnatural
amino acids also increase the likelihood that a peptide may be seen as foreign and
set off undesirable immune reactions, such as human immunogenicity and injection site
reactions.
Fatty acids, through their albumin binding motifs, can improve the
pharmacokinetics of a peptide by extending the half-life, for example. While use of fatty
acids can improve peptide half-life, it was discovered by Applicants as part of the present
invention that the length, composition, and placement of the fatty acid chain and the
linker between the peptide and the fatty acid chain can have unexpected effects on the
balancing of the GIP and GLP-1 agonist activity.
Tolerability of certain GLP- 1 analogues has been found to prevent a dose of the
GLP-1 analogue that reaches better efficacy for glycemic control and weight loss. The
most common side effects assigned to GLP-1 analogues are nausea and vomiting but
some compounds may also impact heart rate. The HPA axis is part of a physiological
stress response and GLP- 1 has been found to stimulate the HPA axis in rats resulting in
increased corticosterone levels providing a potential link to adverse events such as
increased heart rate. As part of the present invention, Applicants unexpectedly found that
a compound of the present invention did not lead to elevated corticosterone levels like
seen with semaglutide in a rat model and so possibly can be dosed to higher efficacy
levels than GLP-1R-selective agents.
There remains a need to provide a compound that is a balanced co-agonist of GIP
and GLP-1 receptors, but is selective against related glucagon and GLP-2 receptors.
Also, there remains a need to provide a compound with balanced co-agonist activity of
GIP and GLP-1 receptors which may provide weight loss given activity found in animal
models. Additionally, there remains a need to provide a compound with balanced coagonist
activity of GIP and GLP- 1 receptors that delivers adequate stability against DPP
IV and other forms of degradation, but while still maintaining a low immunogenicity
potential. Also, there remains a need to provide a compound with balanced co-agonist
activity of GIP and GLP- 1 receptors that supports potential once-weekly dosing in
humans.
Accordingly, certain compounds of the present invention have lower potential for
immunogenicity and injection site reactions than certain GIP-GLP-1 co-agonist
compounds in the art. Certain compounds of the present invention have potential for
producing weight loss in patients based on animal energy expenditure data. Furthermore,
certain compounds of the present invention have a balanced co-agonist activity against
GIP and GLP-1 receptors and selectivity against both glucagon and GLP-2 receptors, low
immunogenicity potential, and pharmacokinetic (PK) characteristics that support onceweekly
dosing in humans.
Accordingly, an embodiment of the present invention provides a compound of
Formula I :
YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS;
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(YGlu)a-CO-(CH2)b-C0 2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe
or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary
amide (SEQ ID NO: 11), or a pharmaceutically acceptable salt thereof.
In a further embodiment, the present invention provides a compound of Formula I,
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through conjugation
to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(YGlu)a-CO-(CH 2)b-C0 2H wherein a is 1 to 2 and b is 10 to 18; X 3 is Phe; and the
C-terminal amino acid is optionally amidated as a C-terminal primary amide, or a
pharmaceutically acceptable salt thereof.
In a further embodiment, the present invention provides a compound of Formula I,
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through conjugation
to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(YGlu)a-CO-(CH 2)b-C0 2H wherein a is 1 to 2 and b is 10 to 18; X 3 is 1-Nal; and
the C-terminal amino acid is optionally amidated as a C-terminal primary amide, or a
pharmaceutically acceptable salt thereof.
In a further embodiment, the present invention provides a compound of Formula I,
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through conjugation
to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-
acetyl) 2-(YGlu)a-CO-(CH 2)b-C0 2H wherein a is 1 to 2 and b is 14 to 18; X 3 is Phe or 1-
Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary amide,
or a pharmaceutically acceptable salt thereof. In a further embodiment, the present
invention provides a compound wherein b is 16 to 18. Additionally, the present invention
provides a compound wherein b is 18.
In a further embodiment, the present invention provides a compound of Formula I,
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through conjugation
to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-
acetyl) 2-(YGlu)a-CO-(CH 2)b-C0 2H wherein a is 1 and b is 10 to 18; X 3 is Phe or 1-Nal;
and the C-terminal amino acid is optionally amidated as a C-terminal primary amide, or a
pharmaceutically acceptable salt thereof.
In a further embodiment, the present invention provides a compound of Formula I,
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through conjugation
to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-
acetyl) 2-(yGlu)a-CO-(CH 2)b-C0 2H wherein a is 2 and b is 10 to 18; X 3 is Phe or 1-Nal;
and the C-terminal amino acid is optionally amidated as a C-terminal primary amide, or a
pharmaceutically acceptable salt thereof.
In a further embodiment, the present invention provides a compound of Formula I,
wherein X is Aib; X2 is Aib; K at position 20 is chemically modified through conjugation
to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(YGlu)a-CO-(CH 2)b-C0 2H wherein a is 1 to 2 and b is 10 to 18; X3 is Phe or 1-
Nal; and the C-terminal amino acid is amidated as a C-terminal primary amide, or a
pharmaceutically acceptable salt thereof.
In an embodiment, the present invention provides a compound of the Formula:
YX1EGTFTSDYSIX 2LDKIAQKAFVQWLIAGGPSSGAPPPS;
wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(YGlu)i-CO-(CH2 )i8 -C0 2H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 3), or a pharmaceutically acceptable salt
thereof.
In an embodiment, the present invention provides a compound of the Formula:
YXiEGTFTSDYSIX 2LDKIAQKAX 3VQWLIAGGPSSGAPPPS;
wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(YGlu)2-CO-(CH 2 )i8 -C0 2H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 4), or a pharmaceutically
acceptable salt thereof.
In an embodiment, the present invention provides a compound of the Formula:
YXiEGTFTSDYSIX 2LDKIAQKAFVQWLIAGGPSSGAPPPS;
wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(YGlu)i-CO-(CH2 )i6 -C0 2H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 5), or a pharmaceutically acceptable salt
thereof.
In an embodiment, the present invention provides a compound of the Formula:
YXiEGTFTSDYSIX 2LDKIAQKAFVQWLIAGGPSSGAPPPS;
wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i6-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 6), or a pharmaceutically acceptable salt
thereof.
In an embodiment, the present invention provides a compound of the Formula:
YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i8-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 7), or a pharmaceutically acceptable salt
thereof.
In an embodiment, the present invention provides a compound of the Formula:
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i6-C02H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 8), or a pharmaceutically
acceptable salt thereof.
In an embodiment, the present invention provides a compound of the Formula:
YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS
wherein X is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i6-C02H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 9), or a pharmaceutically
acceptable salt thereof.
In an embodiment, the present invention provides a compound of the Formula:
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i8-C02H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 10), or a pharmaceutically
acceptable salt thereof.
In an embodiment, the present invention provides a composition comprising a
compound of the present invention with a pharmaceutically acceptable carrier, diluent, or
excipient.
In an embodiment, the present invention provides a method of treating type 2
diabetes mellitus, comprising administering to a patient in need thereof, an effective
amount of a compound of the present invention. In a further embodiment, the present
invention provides a method of treating type 2 diabetes mellitus further comprising
administering simultaneously, separately, or sequentially in combination with an effective
amount of one or more agents selected from metformin, thiazolidinediones, sulfonylureas,
dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters.
In an embodiment, the present invention provides a method to improve glycemic
control in adults with type 2 diabetes mellitus, comprising administering to a patient in
need thereof, an effective amount of a compound of the present invention as an adjunct to
diet and exercise. In an embodiment, the present invention provides a method for chronic
weight management in adults with an initial body mass index > 27 and type 2 diabetes
mellitus, comprising administering to a patient in need thereof, an effective amount of a
compound of the present invention as an adjunct to a reduced-calorie diet and increased
physical activity.
In an embodiment, the present invention provides a method to treat metabolic
syndrome, comprising administering to a patient in need thereof, an effective amount of a
compound of the present invention. In a further embodiment, the present invention
provides a method to treat dyslipidemia, obesity, and/or hepatic steatosis associated with
insulin resistance and diabetes, comprising administering to a patient in need thereof, an
effective amount of a compound of the present invention. Additionally, the present
invention provides a method to treat frailty or increase bone strength, comprising
administering to a patient in need thereof, an effective amount of a compound of the
present invention.
In an embodiment, the present invention provides a compound of the present
invention for use in therapy. In a further embodiment, the present invention provides a
compound of the present invention for use in the treatment of type 2 diabetes mellitus. In
a further embodiment, the present invention provides a compound of the present
invention in simultaneous, separate, or sequential combination with one or more agents
selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4
inhibitors, and sodium glucose co-transporters for use in the treatment of type 2 diabetes
mellitus.
In an embodiment, the present invention provides a compound of the present
invention for use in glycemic control in adults with type 2 diabetes mellitus as an adjunct
to diet and exercise. In an embodiment, the present invention provides a compound of the
present invention for use in chronic weight management in adults with an initial body
mass index > 27 and type 2 diabetes mellitus as an adjunct to a reduced-calorie diet and
increased physical activity.
In an embodiment, the present invention provides the use of a compound of the
present invention for the manufacture of a medicament for the treatment of type 2
diabetes mellitus. In a further embodiment, the present invention provides the use of a
compound of the present invention in simultaneous, separate, or sequential combination
with one or more agents selected from metformin, thiazolidinediones, sulfonylureas,
dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters for the manufacture
of a medicament for the treatment of type 2 diabetes mellitus.
The present invention provides compounds that display a balanced GIP and GLP-
1 activity. Balanced activity against GIP and GLP-1 as used herein refers to a compound
that has affinity for GIP receptors and GLP-1 receptors in an in vitro binding assay at a
molar ratio that is close to 1:1, such as 1:1 GLP-l/GIP, 2:1 GLP-l/GIP, 3:2 GLP-l/GIP,
1:2 GLP-l/GIP, or 2:3 GLP-l/GIP.
The present invention provides compounds that display selectivity for GIP and
GLP-1 receptors versus receptors for glucagon and GLP-2. The term "selectivity" or
"selective against" when used herein to reference GIP and GLP-1 activity in comparison
to glucagon activity, refers to a compound that displays 1000-, 500-, or about 100-fold
higher potency for GIP and GLP-1 over glucagon when the data is normalized from the
respective in vitro binding assays. The term "selectivity" or "selective against" when
used herein to reference GIP and GLP- 1 activity in comparison to GLP-2 activity, refers
to a compound that displays 250-, 200-, 100-, or about 50-fold higher potency for GIP
and GLP-1 over GLP-2 when the data is normalized from the respective in vitro
functional assays.
The present invention provides a method for treatment of type 2 diabetes in a
patient comprising administering to a patient in need of such treatment an effective
amount of a compound of the present invention, or a pharmaceutically acceptable salt
thereof. The present invention also provides a method for treatment of type 2 diabetes in
a patient comprising administering to a patient in need of such treatment an effective
amount of a compound of the present invention, or a pharmaceutically acceptable salt
thereof, wherein the administration is subcutaneous. The present invention also provides
a method of treatment of type 2 diabetes in a patient comprising administering to a patient
in need of such treatment an effective amount of a compound of the present invention, or
a pharmaceutically acceptable salt thereof, and simultaneously, separately, or sequentially
an effective amount of one or more other active ingredients. In one embodiment, the
other active ingredient or ingredients is currently available oral glucose lowering drugs
from a class of drugs that is considered prior to administration the standard of care as
determined by industry guidelines such as the American Diabetes Association.
The compounds of the present invention utilize a fatty acid chemically conjugated
to the epsilon-amino group of a lysine side-chain. The fatty acid is conjugated to the
epsilon-amino group of a lysine side-chain through a linker. The linker comprises [2-(2-
Amino-ethoxy)-ethoxy]-acetyl) 2-(yGlu)a wherein a is 1 to 2. The fatty acid and the
gamma-glutamic acid in the linker act as albumin binders, and provide the potential to
generate long-acting compounds. Compounds of the present invention comprise a lysine
at position 20 that is chemically modified through conjugation to the epsilon-amino group
of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(yGlu) a-CO-(CH2)b-
CO2H wherein a is 1 to 2 and b is 10 to 20. As shown in the chemical structures of
Examples 1 and 2, the first unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl is linked to the
epsilon-amino group of the lysine side-chain. The second unit of [2-(2-Amino-ethoxy)-
ethoxy]- acetyl is then attached to the amino-group of the first unit of [2-(2-Aminoethoxy)-
ethoxy]-acetyl. Then, the first unit of yGlu is attached to the amino -group of the
second unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl through the -carboxyl group of the
side-chain. When a = 2, the second unit of yGlu is attached to the -amino -group of the
first unit of yGlu through the -carboxyl group of the side-chain. Finally, the symmetrical
fatty acid is attached to the -amino -group of the first (when a = 1) or second (when a
=2) unit of yGlu.
The compounds of the invention are preferably formulated as pharmaceutical
compositions administered by parenteral routes (e.g., subcutaneous, intravenous,
intraperitoneal, intramuscular, or transdermal). Such pharmaceutical compositions and
processes for preparing same are well known in the art. (See, e.g., Remington: The
Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition, Lippincott, Williams
& Wilkins, 2006). The preferred route of administration is subcutaneous.
The compounds of the present invention may react with any of a number of
inorganic and organic acids to form pharmaceutically acceptable acid addition salts.
Pharmaceutically acceptable salts and common methodology for preparing them are well
known in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties,
Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011); S.M. Berge, et al,
"Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January
1977. Pharmaceutically acceptable salts of the present invention include trifluoroacetate,
hydrochloride, and acetate salts.
As used herein, the term "effective amount" refers to the amount or dose of
compound of the present invention, or a pharmaceutically acceptable salt thereof which,
upon single or multiple dose administration to the patient, provides the desired effect in
the patient under diagnosis or treatment. An effective amount can be readily determined
by the attending diagnostician, as one skilled in the art, by the use of known techniques
and by observing results obtained under analogous circumstances. In determining the
effective amount for a patient, a number of factors are considered by the attending
diagnostician, including, but not limited to: the species of mammal; its size, age, and
general health; the specific disease or disorder involved; the degree of or involvement or
the severity of the disease or disorder; the response of the individual patient; the particular
compound administered; the mode of administration; the bioavailability characteristics of
the preparation administered; the dose regimen selected; the use of concomitant
medication; and other relevant circumstances.
As used herein, the term "treating" or "to treat" includes restraining, slowing,
stopping, or reversing the progression or severity of an existing symptom or disorder.
As used herein, "semaglutide" refers to a chemically synthesized GLP-1 analogue
that has the peptide backbone and overall compound structure of that found in CAS
Registry Number 910463-68-2.
Certain compounds of the present invention are generally effective over a wide
dosage range. For example, dosages for once-weekly dosing may fall within the range of
about 0.05 to about 30 mg per person per week. Certain compounds of the present
invention may be dosed daily. Additionally, certain compounds of the present invention
may be dosed once-weekly.
The amino acid sequences of the present invention contain the standard single
letter or three letter codes for the twenty naturally occurring amino acids. Additionally,
"Aib" is alpha amino isobutyric acid, and "1-Nal" is 1-Naphthylalanine.
The present invention also encompasses novel intermediates and processes useful
for the synthesis of compounds of the present invention, or a pharmaceutically acceptable
salt thereof. The intermediates and compounds of the present invention may be prepared
by a variety of procedures known in the art. In particular, the process using chemical
synthesis is illustrated in the Examples below. The specific synthetic steps for each of the
routes described may be combined in different ways to prepare compounds of the present
invention, or salts thereof. The reagents and starting materials are readily available to one
of ordinary skill in the art. It is understood that these Examples are not intended to be
limiting to the scope of the invention in any way.
EXAMPLE 1
YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i8-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 3)
trifluoroacetate salt
The above structure contains the standard single letter amino acid code with
exception of residues Aib2, Aibl3 and K20 where the structures of these amino acid
residues have been expanded.
The peptide according to SEQ ID NO: 3 of the present invention is generated by
solid-phase peptide synthesis using a Fmoc/t-Bu strategy carried out on a Symphony
automated peptide synthesizer (PTI Protein Technologies Inc.) starting from RAPP AMRink
Amide resin and with couplings using 6 equivalents of amino acid activated with
diisopropylcarbodiimide (DIC) and hydroxybenzotriazole (HOBt) (1:1:1 molar ratio) in
dimethylformamide (DMF) for 90 min at 25°C.
Extended couplings (4h each) for Pro31, Trp25, Gln24, Val23, Phe22, Lys20,
Gly4, Glu3 and Aib2 are necessary to improve the quality of the crude peptide. A Fmoc-
Lys(Alloc)-OH building block is used for Lys20 coupling (orthogonal protecting group)
to allow for site specific attachment of the fatty acid moiety later on in the synthetic
process. The following conditions are used for the coupling of Fmoc-Ile-OH at position
12: Fmoc-Ile-OH (6 equiv), PyBOP (6 equiv), and DIEA (12 equiv) in DMF for 24 h at
25°C. The N-terminal residue is incorporated as Boc-Tyr(tBu)-OH using DIC-HOBt
protocols as described above.
After finishing the elongation of the peptide-resin described above, the Alloc
protecting group present in Lys20 is removed using catalytic amounts of Pd(PPh3)4 in the
presence of PhSiH3 as a scavenger. Additional coupling/deprotection cycles using a
Fmoc/t-Bu strategy to extend the Lys20 side-chain involved Fmoc-NH-PEG2-CH2COOH
(ChemPep Catalog#280102), Fmoc-Glu(OH)-OtBu (ChemPep Catalog#100703) and
HOOC-(CH2)i8 -COOtBu. In all couplings, 3 equivalents of the building block are used
with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.
Concomitant cleavage from the resin and side chain protecting group removal are
carried out in a solution containing trifluoroacetic acid (TFA): triisopropylsilane : 1,2-
ethanedithiol: water : thioanisole 90:4:2:2:2 (v/v) for 2 h at 25°C followed by
precipitation with cold ether. Crude peptide is purified to > 99% purity (15-20% purified
yield) by reversed-phase HPLC chromatography with water / acetonitrile (containing
0.05% v/v TFA) gradient on a C18 column, where suitable fractions are pooled and
lyophilized.
In a synthesis performed essentially as described above, the purity of Example 1
was examined by analytical reversed-phase HPLC, and identity was confirmed using
LC/MS (observed: M+3H+/3 =1605.2; Calculated M+3H+/3 =1605.5; observed: M+4H+/4
=1204.3; Calculated M+4H+/4 =1204.4).
EXAMPLE 2
YX1EGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(yGlu)2-CO-(CH2)i8 -C0 2H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 4)
trifluoroacetate salt
The above structure contains the standard single letter amino acid code with
exception of residues Aib2, Aibl3, K20 and 1-Nal22 where the structures of these amino
acid residues have been expanded.
The peptide according to SEQ ID NO: 4 of the present invention is synthesized
similarly as described above in Example 1. The following conditions are used for the
coupling of Fmoc-lNal-OH at position 22: Fmoc-lNal-OH (6 equiv), PyBOP (6 equiv),
and DIEA (12 equiv) in DMF for 4 h at 25°C.
EXAMPLE 3
YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i6-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 5)
trifluoroacetate salt
The compound according to SEQ ID NO: 5 of the present invention is synthesized
similarly as described above for Example 1.
EXAMPLE 4
YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i6-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 6)
trifluoroacetate salt
The compound according to SEQ ID NO: 6 of the present invention is synthesized
similarly as described above for Example 1.
EXAMPLE 5
YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i8-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide (SEQ ID NO: 7)
trifluoroacetate salt
The compound according to SEQ ID NO: 7 of the present invention is synthesized
similarly as described above for Example 1.
EXAMPLE 6
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i6-C02H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 8)
trifluoroacetate salt
The compound according to SEQ ID NO: 8 of the present invention is synthesized
similarly as described above for Example 1.
EXAMPLE 7
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(YGlu)2-CO-(CH2 )i6 -C02H; X3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 9)
trifluoroacetate salt
The compound according to SEQ ID NO: 9 of the present invention is synthesized
similarly as described above for Example 1.
EXAMPLE 8
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein Xi is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH 2)i8 -C0 2H; X3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide (SEQ ID NO: 10)
trifluoroacetate salt
The compound according to SEQ ID NO: 10 of the present invention is
synthesized similarly as described above for Example 1.
ASSAYS
Provided below are the conditions and data for Examples in several assays: in
vitro function and selectivity, immunogenicity profiling, pharmacokinetics, and in vivo
type 2 diabetes models.
In vitro function and selectivity
In vitro binding potency to human GLP-1 and GIP Receptors
The in vitro binding potency of compounds of the present invention to human GIP
and GLP-1 receptors is evaluated by measuring the binding affinities, as K;, using crude
cellular membranes obtained from clonal cell lines over-expressing either the human
GLP1R cDNA or human GIP-R cDNA.
The human glucose-dependent insulinotropic polypeptide receptor binding assay
uses hGIP-R (Usdin,T.B., Gruber.C, Modi,W. and Bonner,T.I., GenBank: AAA84418.1)
cloned into pcDNA3.1 (Promega)-NeoR plasmid. The hGIP-R-pcDNA3.1/Neo plasmid
is transfected into Chinese Hamster Ovary cells, CHO-S, for suspension cultures and
selected in the presence of 500 g/mL Geneticin (Invitrogen).
Crude plasma membranes are prepared using cells from suspension culture. The
cells are lysed on ice in hypotonic buffer containing 25 mM Tris HC1, pH 7.5, 1mM
MgCl2, DNAsel, 20 /mL, and Roche Complete™ Inhibitors without EDTA. The cell
suspension is homogenized with a glass dounce homogenizer using a Teflon® pestle for
25 strokes. The homogenate is centrifuged at 4°C at 1800 x g for 15 minutes. The
supernatant is collected and the pellet resuspended in hypotonic buffer and rehomogenized.
The mixture is centrifuged at 1800 x g for 15 minutes. The second
supernatant is combined with the first supernatant. The combined supernatants are recentrifuged
at 1800 x g for 15 minutes to clarify. The clarified supernatant is transferred
to high speed tubes and centrifuged at 25,000 x g for 30 minutes at 4°C. The membrane
pellet is resuspended in homogenization buffer and stored as frozen aliquots at -80 °C
freezer until use.
GIP is radioiodinated by the I-125-lactoperoxidase procedure (Markalonis, J.J.,
Biochem. J. 113:299 (1969)) and purified by reversed phase HPLC (Perkin-Elmer Life
and Analytical Sciences NEX-402). The specific activity is 2200 Ci/mmol. K
determination is performed by homologous competition using cold hGIP instead of
saturation binding. The receptor binding assay is carried out using a Scintillation
Proximity Assay (SPA) with wheat germ agglutinin (WGA) beads (Perkin Elmer Life and
Analytical Sciences) previously blocked with 1% fatty acid free BSA (Gibco, 7.5%
BSA). The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1mM
MgCl2, 0.1% fatty acid free BSA, 0.003% Tween20, and Roche Complete™ Inhibitors
without EDTA. hGIP and the compounds of the present invention are dissolved in 100%
DMSO and stored at -20°C. The compounds are serially diluted into binding buffer.
Next, 10 diluted compound or 100% DMSO is transferred into Corning® 3632 clear
bottom assay plates containing 40 assay binding buffer or cold GIP (NSB at 0.1 
final). Then, 90 membranes (3 g/well), 50 [1 5I] GIP (Perkin Elmer Life and
Analytical Sciences at 0.15 nM final in reaction), and 50 of WGA beads (150 g/well)
is added, sealed, and mixed on a plate shaker for 1 minute. Plates are read with a
MicroBeta® scintillation counter after 12 hours of settling time at room temperature.
Results are calculated as a percentage of specific I-125-GIP binding in the
presence of compound. The Absolute IC50 concentration is derived by non-linear
regression of percent specific binding of I-125-GIP versus the concentration of compound
added. The IC50 concentration is converted to Ki using the Cheng-Prusoff equation.
The GLP-1 receptor binding assay uses cloned human glucagon-like peptide 1
receptor (hGLP-lR) (Graziano MP, Hey PJ, Borkowski D, Chicchi GG, Strader CD,
Biochem Biophys Res Commun. 196(1): 141-6, 1993) isolated from 293HEK membranes.
The hGLP-lR cDNA is subcloned into the expression plasmid phD (Trans-activated
expression of fully gamma-carboxylated recombinant human protein C, an antithrombotic
factor. Grinnell, B.W., Berg, D.T., Walls, J . and Yan, S.B. Bio/Technology 5 : 1189-1192,
1987). This plasmid DNA is transfected into 293HEK cells and selected with 200 g/mL
Hygromycin.
Crude plasma membranes are prepared using cells from suspension culture. The
cells are lysed on ice in hypotonic buffer containing 25 mM Tris HC1, pH 7.5, 1mM
MgCl2, DNAsel, 20 /mL, and Roche Complete™ Inhibitors without EDTA. The cell
suspension is homogenized with a glass dounce homogenizer using a Teflon® pestle for
25 strokes. The homogenate is centrifuged at 4°C at 1800 x g for 15 minutes. The
supernatant is collected and the pellet resuspended in hypotonic buffer and rehomogenized.
The mixture is centrifuged at 1800 x g for 15 minutes. The second
supernatant is combined with the first supernatant. The combined supernatants are
recentrifuged at 1800 x g for 15 minutes to clarify. The clarified supernatant is
transferred to high speed tubes and centrifuged at 25000 x g for 30 minutes at 4°C. The
membrane pellet is resuspended in homogenization buffer and stored as frozen aliquots at
-80 °C freezer until use.
Glucagon-like peptide 1 (GLP-1) is radioiodinated by the I-125-lactoperoxidase
procedure and purified by reversed phase HPLC at Perkin-Elmer Life and Analytical
Sciences (NEX308). The specific activity is 2200 Ci/mmol. K determination is
performed by homologous competition instead of saturation binding due to high propanol
content in the 1-125 GLP-1 material. The KD is estimated to be 0.329 nM and is used to
calculate Ki values for all compounds tested.
The receptor binding assay is carried out using a Scintillation Proximity Assay
(SPA) with wheat germ agglutinin (WGA) beads previously blocked with 1% fatty acid
free BSA (Gibco). The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1
mM MgCl2, 0.1% fatty acid free BSA, 0.003% Tween20, and Roche Complete™
Inhibitors without EDTA. Glucagon-like peptide 1 is dissolved in 100% DMSO at 1
mg/mL and stored frozen at -20 °C in 30 aliquots. The glucagon-like peptide 1
aliquot is diluted and used in binding assays within an hour. The peptide analog is
dissolved in 100% DMSO and serially diluted in 100% DMSO. Next, 10 diluted
compounds of the present invention or 100% DMSO are transferred into Corning® 3632
clear bottom assay plates containing 40 assay binding buffer or cold glucagon (NSB at
1 final). Then, 90 ΐ membranes (0.5 g/well), 50 ΐ 1-125 Glucagon-like peptide 1
(0.15 nM final in reaction), and 50 ΐ of WGA beads (150 g/well) is added, sealed, and
mixed on a plate shaker for 1 minute. Plates are read with a PerkinElmer Life and
Analytical Sciences Trilux MicroBeta® scintillation counter after 12 hours of settling
time at room temperature.
Results are calculated as a percentage of specific I-125-Glueagon-like peptide 1
binding in the presence of compounds. The Absolute IC50 concentration of compound is
derived by non-linear regression of percent specific binding of I-125-Glucagon-like
peptide 1 versus the concentration of compound added. The IC50 concentration is
converted to Ki using the Cheng-Prusoff equation.
In experiments performed essentially as described in this assay, certain
compounds of the present invention display an hGLP-lR/hGIPR ratio of approximately
0.5-4.0 (Table 1). The molar binding ratio is normalized to the corresponding molar ratio
of a mixture of native GIP and GLP-1. This normalization factor is 4.53 based on binding
data for GIP (Ki=0.175 nM) and GLP-1 (Ki=0.793 nM). The value of 1.48 demonstrates
the balanced co-agonist activity of Example 1.
Table 1: Receptor Binding Affinity, Ki, nM (SEM, n)
Means are expressed as geometric means with the standard error of the mean
(SEM) and the number of replicates (n) indicated in parenthesis. A qualifier (>) indicates
data did not reach 50% inhibition and the is calculated using the highest concentration
tested in the assay.
Functional hGIP-R, hGLP-lR, and hGCGR assays.
The in vitro functional activity towards human GIP, GLP-1, and glucagon
receptors for compounds of the present invention are determined in HEK-293 clonal cell
lines expressing these receptors. Each receptor over-expressing cell line is treated with
compounds of the present invention in DMEM (Gibco Cat# 31053) supplemented with
IX GlutaMAX™ (Gibco Cat# 35050), 0.25% FBS, 0.05% fraction V BSA, 250 
IBMX and 20 mM HEPES in a 40 ΐ assay volume. After an incubation of 60 minutes at
room temperature, the resulting increase in intracellular cAMP is quantitatively
determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit (Bedford, MA). Briefly,
cAMP levels within the cell are detected by adding the cAMP-d2 conjugate in cell lysis
buffer (20 ΐ) followed by the antibody anti-cAMP-Eu +-Cryptate, also in cell lysis buffer
(20 ΐ). The resulting competitive assay is incubated for at least 60 minutes at room
temperature, then detected using a PerkinElmer Envision® instrument with excitation at
320 nm and emission at 665 nm and 620 nm. Envision units (emission at
665nm/620nm* 10,000) are inversely proportional to the amount of cAMP present and are
converted to nM cAMP per well using a cAMP standard curve. The amount of cAMP
generated (nM) in each well is converted to a percent of the maximal response observed
with either human GIP(1-42)NH2, human GLP-1(7-36)NH2, or human glucagon controls.
A relative EC5 0 value and percent top (Emax) is derived by non-linear regression analysis
using the percent maximal response versus the concentration of the compound of the
present invention, fitted to a four-parameter logistic equation.
In experiments performed essentially as described in this assay, certain
compounds of the present invention demonstrate activity against human GIP and GLP- 1
receptors, while also demonstrating selectivity over the glucagon receptor. In Table 2,
functional potency against the receptors is shown for the native hGIP(l-42)NH 2, hGLP-
1(7-36)NH2, and hGlucagon controls, and certain compounds of the present invention.
Table 2. Functional Potency (EC50) against human GIP, GLP-1, and glucagon
receptors.
Compound Human GIP-R Human GLP- 1R Human GCGR
EC50, E m •> EC50, Emax EC50, Emax
nM+SEM nM+SEM nM+SEM,
(n) (n) (n)
Example 1 11.0+0.9 97.9+3.0 71.2+7.2 85.2+4.4 >1000 (13) ND
(17) (17)
Example 3 3.15+0.34 106+3 33.9+3.2 96.2+6.2 >1000 (10) ND
(14) (14)
Example 6 4.40+0.71 106+3 28.4+4.2 104+4 >1000 (9) ND
(13) (13)
Example 7 8.07+0.70 106+3 35.5+2.6 97.2+3.4 >1000 (13) ND
(17) (17)
Example 8 21.1+2.7 108+4 57.9+6.8 88.4+2.3 >1000 (10) ND
(14) (13)
Example 4 3.76+0.83 102+3 66.9+14.9 100+5 >1000 (2) ND
(6) (6)
Example 2 17.5+2.0 94.7+2.0 75.7+6.0 98.2+5.5 >1000 (9) ND
(13) (13)
Example 5 8.76+0.86 105+2 70.9+9.7 105+4 >1000 (10) ND
(10) (10)
hGLP-l(7- 0.176 102+2
36)NH2 +0.015
(17)
hGIP(l- 0.135 100+1
42)NH2 +0.010
(17)
hGlucagon 0.0208 115+2
+0.0024
(13)
Means for EC5 0 are expressed as geometric means +/- standard error of the mean
(SEM) with the number of replicates (n) indicated in parenthesis. Means for Emax are
expressed as the arithmetic mean +/- standard error. ND signifies that agonist activity
was not detected. A qualifier (>) indicates that an EC5 0 could not be determined. All
values shown are to three significant digits
Functional activation of hGIP-R cells to generate intracellular cAMP in incretinsecreting
cell lines
The functional activity of hGIP-R for compounds of the present invention are
demonstrated by the ability of the compounds to generate intracellular cAMP in GLUTag
cells, a stable immortalized relatively differentiated murine enteroendocrine cell line that
expresses the proglucagon gene and secretes the glucagon-like peptides in a regulated
manner. The cells are maintained at 37°C, 5% CO2, 95% humidity in DMEM medium
supplemented with 5.5 mM glucose, 10% FBS, and 2 mM glutamine. Prior to assay, cells
are trypsinized, pelleted, and seeded into 96-well tissue culture assay plates at a density of
20,000 cells/well. Cells are allowed to attach and are incubated for 48 hours at 37°C, 5%
CO2. On the day of the assay, media is decanted from cells and 50 ΐ EBSS Buffer (0.1%
BSA, 2 mM glucose and 0.25 mM IBMX) containing a range of compound
concentrations (0.001 - 3 ) is added to cells. The plate is incubated at 37°C for one
hour and cAMP levels determined using Cisbio Dynamic 2 cAMP HTRF kit (Bedford,
MA). 25 ΐ of anti-cAMP cryptate and 25 ΐ cAMP d2 is added to each well and plates
incubated for one hour at room temperature. Plates are read at 620nm and 665nm on a
Tecan Genios Pro. Results are calculated from the 665nm/620nm ratio multiplied by
10000, and converted to nM cAMP per well using a cAMP standard curve. Data is
analyzed with GraphPad using a 4-parameter non-linear logistic algorithm.
In experiments performed essentially as described in this assay, certain
compounds of the present invention show a dose-dependent, enhanced cAMP
accumulation of GLUTag cells (Table 3). The native GLP-1 control fails to induce any
changes in cAMP at all concentrations tested and indicates that this cell system
exclusively expresses the GIP receptor; therefore, certain compounds of the present
invention can be shown to exert an effect through the GIP receptor.
Table 3: EC50 in GLUTag cells.
Compound Average EC50, nM (n)
Example 3 1610 (1)
Example 7 2746 (1)
Example 4 2186 (1)
Example 2 2918 (1)
Example 1 1494 (2)
Native GIP 11.62 (3)
Measurement of Intracellular cAMP in HEK293 Cells Transiently Expressing the
Human GLP-2 Receptor
The functional activity of hGLP-2R in the presence of compounds of the present
invention is demonstrated by measuring intracellular cAMP in HEK293 cells. These cells
are passaged in complete medium, transfected in suspension with Promega Fugene6
reagent and human full-length GLP-2R cDNA in pcDNA3.1 expression vector, and
allowed to adhere to tissue culture flasks in a humidified 37°C 5% CO2 environment.
Following approximately 48 hours of propagation, cells are lifted, strained, and
cryopreserved with controlled rate freezing and 10% DMSO as a cryoprotectant. In
subsequent assays, a single assay-ready vial from the same cell freeze is thawed to
minimize inter-assay variation. On the day of the cellular assay, freezing medium is
exchanged with Invitrogen 31053 DMEM containing 0.5% FBS.
Cells are counted for viability and equilibrated for approximately one to two hours
at 37°C prior to treatment. Compounds of the present invention are solubilized in DMSO
and immediately diluted in DMEM medium containing 0.1% fraction V BSA and the
non-specific phosphodiesterase inhibitor, IBMX. Duration of treatment is 30 minutes at
37°C. Final DMSO concentration does not exceed 1.1%, and final IBMX concentration
is 250 . Cyclic AMP is measured using the dynamic 2 assay with homogenous timeresolved
fluorescence technology (Cisbio Bioassays, Bedford, MA). Respective cAMP
concentrations are deduced from the ratio method of calculation and external standards.
Sigmoidal dose-responses of tested compounds are examined using the four parameter
logistic equation and compared to the native Cis -acylated ligand.
In experiments performed essentially as described in this assay, the Cis -acylated
human GLP-2 control has an EC5 0 value for receptor activation of 1.71 nM while certain
compounds of the present invention have EC5 0 values from approximately lOOx to lOOOx
higher. The EC5 0 values for certain compounds of the present invention demonstrate
selectivity against the GLP-2 receptor.
Table 4: GLP-2R functional activity measurement in HEK293 cells.
Rodent islet insulin secretion
To assess action of compounds of the present invention in a system representing
physiological GLP-1R and GIP-R expression levels insulin secretion, compounds are
tested for effects on insulin secretion from wild-type rodent islets.
After common bile duct cannulation in male C57B1/6 mice (22-26 g) or male
Sprague-Dawley rats (approx. 250 g), the pancreas is distended with Hank's buffer (3 ml
for mice or 10 ml for rats, containing 2% BSA and 0.75 mg/ml Clzyme collagenase
(VitaCyte). Subsequently, tissues are digested in Hank's buffer at 37°C for 11-13
minutes (mice) or 14-16 minutes for rat pancreata. Purified islets (Histopaque-1 100
gradient [Sigma-Aldrich], 18 min at 750x gravity) are cultured overnight in RPMI-1640
medium (Invitrogen) containing 10% FBS, 100 U/ml penicillin and 100 g/ml
streptomycin, and preconditioned by starvation in Earle' s Balanced Salt Solution (EBSS)
supplemented with 0.1% BSA and 2.8 mM glucose. Subsequently, islets are incubated in
EBSS (Invitrogen) supplemented with 0.1% BSA, 2.8-11.2 mM glucose and increasing
levels of compound (6 batches of 4 islets/condition). GLP-l(7-36)amide (30 nM) is used
as a positive control. Insulin is measured over 90 minutes in supernatant using the MSD
Insulin Assay (Meso Scale, Gaithersburg, MD).
Certain compounds of the present invention dose-dependently increase insulin
secretion from both rat and mouse islets as depicted in Table 5.
Table 5. Rodent Islet Insulin Secretion
Rat islet insulin secretion
Compound Mean ED50 (nM) N
Example 3 34.9 2
Example 1 15.5 3
Mouse islet insulin secretion
Compound Mean ED50 (nM) N
Example 3 58.9 2
Example 6 51.4 1
Example 7 11.3 1
Example 4 3.5 1
Example 2 30.0 1
Example 1 47.2 2
Immunogenicity Profiling
The risk of immunogenicity for compounds of the present invention is assessed
using in silico prediction programs, such as Epivax in silico analysis. The risk of
immunogenicity for compounds of the present invention is also assessed by an ex-vivo
method to measure cultured T cells responses ( H-thymidine uptake and IL-2 cytokine
secretion) in the presence of compounds of the present invention.
With Epivax immune-informatic tools, an in silico assessment is performed on
compounds of the present invention to predict an immune response following
administration. The analysis utilizes the probability of a 9-mer frame to bind to a given
human leukocyte antigen leukocyte antigen (HLA) allele and then detection of these Epi-
Bars. For Example 1, a EpiMatrix score of approximately +1.13 indicates a much lower
potential to induce an immune response compared to native GIP peptide backbone with a
EpiMatrix score of + 15.4. A GIP / GLP-1 co-agonist Example from WO 2011/119657
had a score of + 29.5.
A measure of predicted clinical immunogenicity is also examined for compounds
of the present invention by using the characterization of CD4+ T-cell proliferation and
IL-2 cytokine secretion in a cohort of 50 healthy donors representative of the world HLA
allotype population. Certain compounds of the present invention demonstrate a degree of
T-cell stimulation and IL-2 secretion following exposure that does not exceed the
threshold associated with known or positive immunogenic compounds, indicating a low
risk of producing clinical immunogenicity.
Pharmacokinetics
Pharmacokinetics in Cynomolgus Monkeys.
The in vivo pharmacokinetic properties for compounds of the present invention
are demonstrated using cynomolgus monkeys. The compounds are administered by a
single intravenous or subcutaneous dose (0.2 mg/kg) in 20 mM citrate buffer (pH 7.0) at a
volume of 0.21 ml/kg. Blood is collected from each animal at 2, 4, 8, 12, 24, 48, 72, 96,
120, 144, 168, 204, 240, and 312 hours post-dosage. The plasma concentrations of
compounds of the present invention are determined by a LC/MS method. Briefly, a
compound of the present invention is extracted from 100% Monkey plasma sample (50
ΐ ) diluted with IX PBS (150 ΐ ) and mixed with N-butanol (400 ΐ ). Three distinct
liquid layers are formed with the compound located in the top layer. A volume of 200 ΐ
is transferred to a v-bottom 96-well plate, dried down using heated Nitrogen gas and
reconstituted with 100 ΐ of 30% acetonitrile/ 0.1% formic acid. 20 ΐ of the
reconstituted sample is injected onto a Supelco Analytical Discovery bio wide C5 3 
column. The column effluent is directed into a Thermo Q-Exactive mass spectrometer for
detection and quantitation.
In experiments performed essentially as described for this assay, Example 1
reached a mean maximum plasma concentration approximately 8 hours post the
subcutaneous dose. The mean half-life is 55 hours and the mean clearance is 0.73
mL/hr/kg. The bioavailability is approximately 83%. This data supports the potential of
once-weekly dosing for Example 1. Data for other compounds of the present invention
are summarized in Table 6.
Table 6: Mean Pharmacokinetic Parameters Following a Single Subcutaneous Dose
of 0.2 mg/kg to Male Cynomolgus Monkeys
Compound Mean Mean Mean Cmax Mean AUC0-inf Mean CL/F
T /2(hr) Tmax (hr) ^g/mL) (m g L) (mL/hr/kg)
Example 3 34 8 3.0 153 1.3
Example 7 3 1 6 2.9 136 1.5
Example 6 23 4 2.2 72.3 2.8
Example 8 23 10 1.0 42.8 4.7
Example 2 43 24 2.1 173 1.2
n = 2, AUCo-inf = area under the curve from 0 to infinity, CL/F = clearance /
bioavailability, Tmax = time to maximum concentration, Cmax = maximum plasma
concentration, Ti 2 = half-life.
Dose potency projection
The intravenous glucose tolerance test (ivGTT) in the rat is used to estimate
relative potency of compounds of the present invention in comparison to semaglutide.
Single subcutaneous (SC) doses of 0.1-10 nmol/kg of each compound are administered to
the rats and an ivGTT is administered to each rat 16 hours post-dosage. Exposure is
measured at the time of the ivGTT, and for exposure response modeling, the insulin AUC
in response to the ivGTT is used as the primary endpoint.
An Emax model is used to compare the exposure response profiles for Example 1 to
semaglutide. In experiments performed essentially as described for this assay, exposure
is essentially the same for Example 1 and semaglutide for the dose levels that had drug
levels above the limit of quantitation of the assay. Both data sets are fit simultaneously
and Eo and Emax values are constrained to be the same for both compounds. Only ED 0
values are fit separately for the compounds. ED50 value for semaglutide is estimated as
0.6 +/- 0.2 nmol/kg. Potency of Example 1 is estimated as a relative potency to
semaglutide, and is 1.7 +/- 0.6 times the potency of semaglutide. Adjusting for CL/F
(apparent clearance) differences between the two molecules in monkeys and also for the
difference in molecular weight, the projected mean human equivalent dose to 1mg
semaglutide is approximately 1.3 mg/week for Example 1.
Type 2 Diabetes
Rat in vivo insulin secretion following intravenous glucose (IVGTT)
Male Wistar rats (Harlan Labs, Indianapolis, IN) are randomized by body weight
and dosed 1.5 ml/kg s.c. 16 hour prior to glucose administration and then fasted. Doses
are vehicle, 0.1, 0.3, 1, 3 and 10 nmol/kg. Animals are weighed, and then anesthetized
with sodium pentobarbital (Nembutal Sodium solution; Ovation Pharmaceuticals) dosed
i.p. (65 mg/kg, 30 mg/ml). A time zero blood sample is collected into EDTA tubes after
which glucose is administered (0.5 mg/kg, 5 ml/kg). Blood samples are collected at 2, 4,
6, 10, 20, and 30 minutes post glucose. Plasma glucose levels are determined using a
Hitachi analyzer (Roche) and plasma insulin is measured by the MSD insulin assay
(Meso Scale, Gaithersburg, MD).
As shown in Table 7, certain compounds of the present invention dosedependently
enhance insulin secretion following i.v. injection of glucose. The ED 0 for
insulin and the maximal increases in insulin secretion (measured as area under the insulin
curve) are given in Table 7.
Table 7. Enhancement of insulin secretion in the rat IVGTT assay
Effect on weight loss, body composition and hepatic steatosis in diet-induced obese
(DIO) mice
The effects on weight loss, body composition and hepatic steatosis in DIO mice
for compounds of the present invention are evaluated in C57/BL6 DIO mice. These
animals, although not diabetic, display insulin resistance, dyslipidemia, and hepatic
steatosis, which are all characteristics of metabolic syndrome, after being placed on a
high fat (60% Kcal from fat) diet for 12 weeks.
In this study, 23-24 week old male diet-induced obese (DIO) C57/B16 male mice
are used, with each weighing 41-49 g and having an initial fat mass ranging from 10.5-
17.5 g. Animals are individually housed in a temperature-controlled (24°C) facility with
a 12 hour light/dark cycle (lights on 22:00), and free access to food and water. After 2
weeks acclimation to the facility, the mice are randomized to treatment groups
(n=5/group) based on body weight so each group has similar starting mean body weight.
Vehicle control, compounds of the present invention (with dose ranging from 10
to 100 nmol/kg), or a long-acting GLPl analogue semaglutide (30 nmol/kg), dissolved in
vehicle (20 mM Citrate Buffer at pH 7.0), are administered by SC injection to ad libitum
fed DIO mice 30-90 minutes prior to the onset of the dark cycle every three days for 15
days. SC injections are made on Day 1, 4, 7, 10, and 13. Daily body weight and food
intake are measured throughout the study. Absolute changes in body weight are
calculated by subtracting the body weight of the same animal prior to the first injection of
compound. On days 0 and 14, total fat mass is measured by nuclear magnetic resonance
(NMR) using an Echo Medical System (Houston, TX) instrument.
On Day 15, blood glucose is measured with Accu-Chek glucometer (Roche) from
tail vein blood and then animals may be sacrificed and livers removed and frozen. Liver
triglycerides, determined from homogenates of livers collected at sacrifice, and plasma
cholesterol are measured on a Hitachi Modular P clinical analyzer. Statistical
comparisons between groups are done using one-way ANOVA followed by Dunnett's
multiple comparison test. The ED 0 for weight loss lowering is determined in GraphPad
Prism using the non-linear fit tool.
In experiments performed essentially as described in this assay, certain
compounds of the present invention reduced body weight and fat mass in a dosedependent
manner (Table 8-13), and compared to semaglutide, may be 3-5x more
efficacious in lowering body weight. The ED50 of Example 1 in percent body weight loss
is 5.422 nmol/kg (95% Confidence interval levels [nmol/kg] = 2.2 to 13.6). Reduced
body weight is found to be primarily due to reduction in fat mass.
Table 8. Percent body weight or fat mass change in DIO mice.
Treatment Dose (nmol/kg) %Change from %Change from
starting body weight starting fat mass
Control 0 -3.14 + 0.88 -4.84 + 1.79
Semaglutide 10 -12.36 + 1.00**** -18.21 + 2.24**
Semaglutide 30 -14.20 + 1.01**** -21.90 + 2.07***
Semaglutide 100 -19.30 + 1.38**** -33.51 + 3.30***
Example 3 10 -13.38 + 0.88**** -20.76 + 2.42***
Example 3 30 -18.13 + 1.44**** -30.90 + 2.06****
Example 3 100 -25.84 + 1.93**** -45.92 + 2.15****
Example 6 10 -15.31 + 1.25**** -24.75 + 1.89****
Example 6 30 -21.62 + 0.92**** -36.30 + 2.47****
Example 6 100 -33.95 + 1.93**** -64.64 + 4.04****
**p<0.01, ***p<0.001, ****p<0.0001 from control group (One-Way ANOVA,
Dunnett' s). The results are expressed as Mean + SEM of 5 mice per group.
Table 9. Percent body weight or fat mass change in DIO mice.
Treatment Dose (nmol/kg) %Change from %Change from
starting body weight starting fat mass
Control 0 -0.74 + 1.49 3.04 + 3.65
Semaglutide 30 -17.03 + 0.98**** -35.94 + 4.09****
Example 2 10 -23.27 + 1.72**** -49.89 + 5.62****
Example 2 30 33.07 + 1.65**** -72.80 + 4.04****
Example 2 100 -34.66 + 1.80**** -76.20 + 3.78****
Example 5 10 -23.42 + 1.43**** -51.28 + 1.89****
Example 5 30 -26.84 + 3.14**** -62.77 + 5.49****
Example 5 100 -37.86 + 2.25**** -81.08 + 1.68****
Example 1 10 -25.18 + .1.82**** -50.98 + 2.87****
Example 1 30 -26.58 + 2.49**** -59.98 + 6.60****
Example 1 100 -38.14 + 1.67**** -79.79 + 3.10****
****p<0.0001 from control group (One-Way ANOVA, Dunnett's). The results are
expressed as Mean + SEM of 5 mice per group.
Table 10. Percent body weight or fat mass change in DIO mice.
****p<0.001 from control group (One-Way ANOVA, Dunnett's). The results are
expressed as Mean + SEM of 5 mice per group.
Table 11. Blood glucose, plasma cholesterol and plasma triglycerides in DIO mice.
Treatment Dose Blood Glucose Plasma Plasma
(nmol/kg) (mg/dl) Cholesterol Triglycerides
(mg/dl) (mg/dl)
Control 0 141.6 + 5.59 303.2 + 13.97 54.2 + 11.14
Semaglutide 10 147.6 + 6.13 226.8 + 13.86** 27.36 + 3.56*
Semaglutide 30 146.8 + 8.43 229.8 + 10.96** 27.9 + 6.01*
Semaglutide 100 134.3 + 9.22 218.4 + 18.70** 36.46 + 5.34
Example 3 10 109.5 + 2.35*** 213.2 + 15.54*** 30.38 + 8.23
Example 3 30 107.6 + 1.32*** 177.4 + 16.58**** 21.32 + 2.48**
Example 3 100 102.00 +0.50**** 194.00 + 14.40*** 20.55 + 4.60**
Example 6 10 105.8 + 2.10*** 198.4 + 6.76**** 20.78 + 4.40**
Example 6 30 100.1 + 3.29**** 186.4 + 17.04**** 26.12 + 6.85*
Example 6 100 103.6 + 3.20**** 151.4 + 14.32**** 17.26 +
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from control group (One-Way
ANOVA, Dunnett's). The results are expressed as Mean + SEM of 5 mice per group.
Table 12. Blood glucose, plasma cholesterol and liver triglycerides in DIO mice.
Treatment Dose Blood Glucose Plasma Liver
(nmol/kg) (mg/dl) Cholesterol Triglycerides
(mg/dl) (mg/g tissue)
Control 0 144.30 + 8.16 233.6 + 12.99 206.65 + 29.47
Semaglutide 30 136.3 + 3.81 161.0 +13.92*** 67.63 + 23.40****
Example 2 10 110.8 + 3.87** 121.8 + 60.77 + 13.24****
13 54****
Example 2 30 110.8 + 3.20** 114.00 + 65.78 + 17.07****
7 ****
Example 2 100 113.2 + 4.86** 109.4 + 56.74 + 17.76****
g3****
Example 5 10 111.00 + 6.56** 126.6 + 48.30 + 8.14****
g7****
Example 5 30 104.5 + 5.30*** 108.2 + 39.60 + 4.71****
13 84****
Example 5 100 105.3 + 6.16*** 108.6 + 67.96 + 13.53****
4 83****
Example 1 10 102.3 + 120.6 + 60.74 + 5.33****
**** 8.55****
Example 1 30 110.7 + 5.85** 118.2 + 45.24 + 5.87****
10 11****
Example 1 100 106.7 + 7.33*** 107.6 + 66.98 + 17.29****
10 43****
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from control group (One-Way
ANOVA, Dunnett's). The results are expressed as Mean + SEM of 5 mice per group.
Table 13. Blood glucose and plasma cholesterol in DIO mice.
Treatment Dose (nmol/kg) Blood Glucose Plasma Cholesterol
(mg/dl) (mg/dl)
Control 0 152.4 + 3.63 243.6 + 13.12
Example 6 10 121.4 + 2.74*** 167.8 + 15.59****
Example 6 30 121.9 + 6.65** 159.8 + 9.99****
Example 6 100 116.1 + 4.67**** 144.2 + 7.12****
Example 7 10 113.6 + 4.16**** 161.8 + 6.2****
Example 7 30 114 + 4_7Q**** 153.6 + 13.47****
Example 7 100 114 + 2.36**** 145.4 + 9.48****
Example 8 10 114.7 + 4.61**** 158.8 + 7.57****
Example 8 30 117.1 + 8.26*** 139.4 + 6.83****
Example 8 100 125.4 + 6.30** 127.8 + 6.34****
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from control group (One- ay
ANOVA, Dunnett's). The results are expressed as Mean + SEM of 5 mice per group.
The effect on energy metabolism in DIO mice
The effects on energy metabolism in DIO mice for compounds of the present
invention are evaluated in 26 week old C57/B16 DIO male mice, weighing 43-50 g. Mice
are individually housed in a temperature-controlled (24°C) facility with a 12 hour
light/dark cycle (lights on 22:00), and with free access to food TD95217 (Teklad) and
water. After 2 weeks acclimation to the facility, mice are randomized to treatment groups
(n=6/group) based on body weight so each group has similar starting mean body weight.
Animals are placed in a PhenoMaster/LabMaster calorimeter (TSE Systems, Chesterfield,
MO) for 3 days of acclimation. Vehicle control (20 mMcitrate buffer at pH 7.0, 10
ml/kg), compounds of the present invention, or a long-acting GLP1 analogue,
semaglutide, (30 nmol/kg) are subcutaneously administered to ad libitum fed DIO mice
30-90 minutes prior to the onset of the dark cycle every three days for 22 days. Heat and
respiratory quotient (RER) are measured by indirect calorimetry as described using an
open-circuit calorimetry system. RER is the ratio of the volume of CO2 produced (VCO2)
to the volume of O 2 consumed (V02). Heat is calculated with full body weight considered:
V02= FlowML*(Vl+V2)/N2Ref*Animal weight*100)
VC02=FlowML *dC02/Animal weight *100)
Heat= (CV02 *V02 +CVC02 *VC02)/l 000;
where CV02=3.941; CVCO2=1.106
In experiments performed essentially as described in this assay, mice treated with
Example 1 significantly increased their metabolic rate 10 to 15 % compared to the control
group, starting from week 2, and sustained the effect throughout the treatment period.
Semaglutide, however, had no effect on metabolic rate. The increase in metabolic rate for
Example 1 partially accounts for the additional weight loss observed with Example 1
treatment in comparison with semaglutide treatment.
The effect on gastric emptying in DIO mice
The effects on gastric emptying in DIO mice for compounds of the present
invention are evaluated in 23 week old diet-induced obese (DIO) male mice (Harlan).
The mice are fasted for 16-17 hours. During the start of fasting period the mice are dosed
subcutaneously with vehicle control (20 mM citrate buffer at pH 7.0); escalating doses of
compounds of the present invention (3, 10, 30 and 100 nmol/kg), or a long-acting GLP1
analogue, semaglutide, (30 nmol/kg). The next day, mice are administered 0.5 ml (0.5
gram) of freshly prepared semi-liquid diet (2 minutes apart) by oral gavage. Water is
removed at this time to prevent dilution of administered diet. Two hours after diet
administration, mice are euthanized two minutes apart by CO2 gas. The stomach is
removed while clamped at both cardiac and pyloric openings, then clamps removed and
the full stomach weighed in a weigh boat. The stomach is then incised and contents
removed. The stomach is washed and dried and re-weighed to assess food contents in
stomach. The % gastric emptying equals 100 x (1 - (food remaining in stomach/food
orally administered)).
In experiments performed essentially as described in this assay, Example 1 slowed
the gastric emptying rate of the semi-liquid diet in a dose dependent manner. The
maximum inhibition of gastric emptying was observed at a dose of 10 nmol/kg +/- dose
(Table 14).
Table 14. Gastric emptying of a semi-liquid diet in lean C57/BL6 DIO mice.
Statistical comparisons between groups are done by using one-way ANOVA
followed by Dunnett's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001 from control group. The results are expressed as mean +/- SEM of 4-5
mice per group.
Plasma Corticosterone Measurements in Sprague Da ley Rats
As suggested in certain published studies, elevated plasma corticosterone levels
are an indicator of possible reduced tolerability for GIP and GLP-1 analogues. Plasma
corticosterone levels are evaluated using Sprague Dawley Rats (Harlan, Indianapolis),
weighing approximately 220 g and acclimated for at least 72 hours before handling. The
rats are then dosed with vehicle (20 mM citrate buffer, pH 7), semaglutide (10 nmol/kg),
or compounds of the present invention at 3, 10 or 30 nmol/kg s.c. with 8 rats per dose
group. The rats are decapitated 16 hours later. Blood is collected into EDTA tubes on
ice, then centrifuged 5 minutes at 8000 RPM in a Eppendorf 5402 tabletop centrifuge.
Plasma is stored at -80°C until analysis.
For corticosterone analysis, corticosterone standards (Sigma, 27840) are prepared
by serial dilutions in HPLC-grade methanol, ¾ 0 and the addition of 5% charcoal
stripped rat serum (Bioreclamation, RATSRM-STRPD-HEV). Rat plasma samples are
diluted with PBS, precipitated with cold methanol, incubated for 20 minutes at -20° C,
and then centrifuged at 14,000 RPM with an Eppendorf 5417R at 4°C. Supernatants are
extracted, evaporated under a stream of N 2 gas, and reconstituted in MeOH/f^O (1:1)
solution. Samples are analyzed on the LC/MS equipped with a XSelect CSH C18 3.5 
HPLC column (2.1 mm x 30 mm) (Waters #186005254).
In experiments performed essentially as described for this assay, Example 1
demonstrated no increase in plasma corticosterone levels at any of the doses tested while
semaglutide had an approximately 4x increase over control.
Table 15: Plasma corticosterone analysis in Sprague Dawley Rats
Corticosterone (ng/ml)
Compound Mean SEM
Vehicle 60.78 8.41
10 nmol/kg Semaglutide 274.57 42.06
3 nmol/kg Example 1 52.21 19.39
10 nmol/kg Example 1 32.46 9.78
nmol/kg Example 1 31.35 5.86
Amino Acid Sequences
SEQ ID NO: 1 (Human GIP)
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ
SEQ ID NO: 2 (Human GLP-1)
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR
SEQ ID NO: 3
YX1EGTFTSDYSIX 2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i8-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide.
SEQ ID NO: 4
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(YGlu)2-CO-(CH2 )i8 -C02H; X3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide.
SEQ ID NO: 5
YX1EGTFTSDYSIX 2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein Xi is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)i-CO-(CH2)i6-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide.
SEQ ID NO: 6
YX1EGTFTSDYSIX 2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i6-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide.
SEQ ID NO: 7
YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i8-C02H; and the C-terminal amino acid is amidated
as a C-terminal primary amide.
SEQ ID NO: 8
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu) i-CO-(CH2)i6-C02H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide.
SEQ ID NO: 9
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(yGlu)2-CO-(CH2)i6-C02H; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide.
SEQ ID NO: 10
YX1EGTFTSDYSIX2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein X i is Aib; X 2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(yGlu)i-CO-(CH 2)i8 -C0 2H ; X 3 is 1-Nal; and the C-terminal amino acid
is amidated as a C-terminal primary amide.
SEQ ID NO: 11
YX1EGTFTSDYSIX 2LDKIAQKAX 3VQWLIAGGPSSGAPPPS
wherein X i is Aib; X2 is Aib; K at position 20 is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-
ethoxy]-acetyl) 2-(YGlu)a-CO-(CH 2)b-C0 2H wherein a is 1 to 2 and b is 10 to 20; X3 is Phe
or 1-Nal; and the C-terminal amino acid is optionally amidated as a C-terminal primary
amide.
AIM:
A compound of the Formula:
YXiEGTFTSDYSIX2LDKIAQKAX3VQWLIAGGPSSGAPPPS;
wherein
Xi is Aib;
X2 is Aib;
K at position 20 is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with ([2-(2-Aminoethoxy)-
ethoxy]-acetyl) 2-(yGlu)a-CO-(CH2)b-C0 2H wherein a is 1
to 2 and b is 10 to 20;
X3 is Phe or 1-Nal;
and the C-terminal amino acid is optionally amidated as a Cterminal
primary amide (SEQ ID NO: 11),
or a pharmaceutically acceptable salt thereof.
The compound of Claim 1, wherein X 3 is Phe.
The compound of Claim 1, wherein X 3 is 1-Nal.
The compound of any one of Claims 1-3, wherein b is 14 to 18.
The compound of Claim 4, wherein b is 16 to 18.
The compound of Claim 5, wherein b is 18.
The compound of any one of Claims 1-6, wherein a is 1.
The compound of any one of Claims 1-6, wherein a is 2.
The compound of any one of Claims 1-8, wherein the C-terminal amino
acid is amidated as a C-terminal primary amide.
The compound of Claim 1, wherein
Xi is Aib
X2 is Aib;
K at position 20 is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with ([2-(2-Aminoethoxy)-
ethoxy]-acetyl) 2-(YGlu)i-CO-(CH2 )i8 -C0 2H;
X3 is Phe;
and the C-terminal amino acid is amidated as a C-terminal primary
amide (SEQ ID NO: 3),
or a pharmaceutically acceptable salt thereof.
11. The compound of Claim 1, wherein
X is Aib
X2 is Aib;
K at position 20 is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with ([2-(2-Aminoethoxy)-
ethoxy]-acetyl) 2-(YGlu)2-CO-(CH2)i8-C02H;
X3 is 1-Nal;
and the C-terminal amino acid is amidated as a C-terminal primary
amide (SEQ ID NO: 4),
or a pharmaceutically acceptable salt thereof.
12. The compound of Claim 1, wherein the Formula is:
The compound of Claiml, wherein the Formula is:
A pharmaceutical composition comprising the compound of any one of
Claims 1-13 with a pharmaceutically acceptable carrier, diluent, or
excipient.
A method of treating type 2 diabetes mellitus, comprising administering to
a patient in need thereof, an effective amount of the compound of any one
of Claims 1-13.
The method of Claim 13, further comprising administering simultaneously,
separately, or sequentially in combination with an effective amount of one
or more agents selected from metformin, thiazolidinediones, sulfonylureas,
dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters.
The compound of any one of Claims 1-13, for use in therapy.
The compound of any one of Claims 1-13, for use in the treatment of type
2 diabetes mellitus.
The compound of any one of Claims 1-13 in simultaneous, separate, or
sequential combination with one or more agents selected from metformin,
thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and
sodium glucose co-transporters.