NOVEL COMPOUND FOR TREATMENT OF SEVERE HYPOGLYCEMIA
The present invention relates to a compound with improved solubility and
physical and chemical stabilities over human glucagon for use in treating diabetes and/or
obesity.
Human glucagon, which has the following amino acid sequence: His-Ser-Gln-
Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-
Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1), is a 29 amino acid peptide hormone
produced in the pancreas. When blood glucose begins to fall, glucagon signals the liver
to break down stored glycogen into glucose for release into the bloodstream, causing
blood glucose level to rise.
In a subject with diabetes, hypoglycemia can arise as a side effect of diabetes
treatment. In addition, the natural glucagon response to hypoglycemia in diabetics may
be impaired, making it harder for glucose levels to return to the normal range. If left
untreated, severe or acute hypoglycemia can cause serious issues such as seizures,
unconsciousness, brain damage, or even death.
Administration of glucagon is an established therapy for treating acute
hypoglycemia. Emergency glucagon administration can restore normal glucose levels
within minutes of administration. Glucagon prepared for administration, however, has
several problems. In aqueous buffers at or near physiological pH, glucagon has poor
solubility. When formulated at low or high pH, glucagon also demonstrates poor
chemical stability and poor physical stability such as gelation and soluble aggregate
formation. To minimize these problems, current commercial glucagon products are
provided as a lyophilized powder with instructions to reconstitute at the time of
administration. In an emergency situation, reconstituting a lyophilized powder is
burdensome and inconvenient. Thus, it is desirable to provide a compound for
therapeutic use that maintains the biological performance of human glucagon under
physiological conditions while also exhibiting sufficient aqueous solubility, chemical
stability and physical stability under non-physiological conditions.
Glucagon analogs with amino acid substitutions to improve solubility and stability
in acidic and physiological pH buffers are disclosed in WO2008086086. There is still a
need for a compound that maintains the biological performance of human glucagon under
physiological conditions while also exhibiting sufficient solubility and chemical and
physical stabilities under non-physiological conditions.
Accordingly, the present invention seeks to provide a compound which maintains
wild-type glucagon activity but also exhibit sufficient solubility as well as chemical and
physical stability. The present invention also provides a compound which is suitable for
pump and/or emergency administration. In addition, the present invention provides a
compound that can be administered in combination with a fast-acting insulin analog in a
dual-chamber pump to provide closed-loop glycemic control.
The present invention provides a compound comprising the amino acid sequence
Tyr-Ser-His-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Lys-Tyr-Leu-Asp-(Aib)-Lys-Lys-
Ala-Ala-Glu-Phe-Val-Ala-Trp-Leu-Leu-Glu-Glu (SEQ ID NO: 2). The present invention
also provides a compound consisting of the amino acid sequence
Tyr-Ser-His-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Lys-Tyr-Leu-Asp-(Aib)-Lys-Lys-
Ala-Ala-Glu-Phe-Val-Ala-Trp-Leu-Leu-Glu-Glu (SEQ ID NO: 2). Unexpectedly, it has
been found that the compound of the present invention exhibits increased aqueous
solubility, increased chemical stability, and reduced fibrillation as compared to human
glucagon in aqueous solution. In addition, the compound of the present invention
demonstrates enhanced solubility at pH near 8. The compound of the present invention
also provides similar activity as human glucagon - e.g., potency, time of action, and
selectivity at the glucagon receptor as compared to human glucagon. Thus, the
compound of the present invention is suitable to treat hypoglycemia, including severe or
acute hypoglycemia. The improved properties of the compound of the present invention
also allow for the preparation of glucagon in aqueous solutions for pump administration
and severe hypoglycemia treatment.
The present invention further provides a method of treating hypoglycemia in a
subject comprising administering a compound comprising the amino acid sequence of
SEQ ID NO: 2. The present invention also provides a method of treating hypoglycemia
in a subject comprising administering a compound consisting of the amino acid sequence
of SEQ ID NO:2. The present invention a further provides a compound comprising the
amino acid sequence of SEQ ID NO: 2 for use in therapy. The present invention also
provides a compound consisting of the amino acid sequence of SEQ ID NO: 2 for use in
therapy. The present invention also provides a compound comprising the amino acid
sequence of SEQ ID NO: 2 for use in the treatment of hypoglycemia. The present
invention also provides a compound consisting of the amino acid sequence of SEQ ID
NO: 2 for use in the treatment of hypoglycemia. The present invention provides a
compound comprising the amino acid sequence of SEQ ID NO: 2 for use in the
manufacture of a medicament for the treatment of hypoglycemia. The present invention
also provides a compound consisting of the amino acid sequence of SEQ ID NO: 2 for
use in the manufacture of a medicament for the treatment of hypoglycemia.
The present invention provides a pharmaceutical composition comprising a
compound comprising an amino acid sequence of SEQ ID NO: 2 and a pharmaceutically
acceptable buffer. The present invention also provides a pharmaceutical composition
comprising a compound consisting of the amino acid sequence of SEQ ID NO: 2 and a
pharmaceutically acceptable buffer. The pharmaceutical composition is preferably an
aqueous solution. As used herein, the term "pharmaceutically acceptable buffer" is
understood to encompass any of the standard pharmaceutical buffers known to those
skilled in the art. Pharmaceutically acceptable buffers for parenteral administration
include, for example, physiological saline, phosphate-buffered saline, citrate -buffered
saline, tris-buffered saline and histidine-buffered saline. Standard pharmaceutical
formulation techniques may be employed such as those described in Remington' s
Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Standard
pharmaceutical formulation techniques may be employed such as those described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
The compound of the present invention can be administered using any standard
route of administration, such as parenterally, intravenously, subcutaneously,
intramuscularly, or transdermally. In an embodiment, the compound of the present
invention is administered subcutaneously or intramuscularly.
The pharmaceutical composition can have a pH that is physiologically acceptable.
In an embodiment, the pharmaceutical composition can have a pH ranging from about 4
to about 8. More preferably, the pharmaceutical composition can have a pH of about 8.
A dose for the compound of the present invention can range from about 0.01
mg to about 100 mg. The dose can range from about 0.01 mg to about 10 mg. The dose
can also range from about 0.1 mg to about 3 mg. In addition, the dose can range from
about 0.01 mg to about 0.03 mg.
The compound of the present invention can be provided as part of a kit. In an
embodiment, the kit is provided with a device for administering the compound to a
human subject. More preferably, the kit comprises a syringe and needle for administering
the compound. Most preferably, the compound is pre-formulated in aqueous solution
within the syringe.
The compound of the present invention can also be used in a pump system, such
as an insulin pump or a bi-hormonal (e.g., insulin-glucagon) pump system.
As used herein, the term "effective amount" or "therapeutically effective amount"
is understood to mean an amount that produces a desired therapeutic effect without
causing unacceptable side effects when administered to a subject. For example, an
"effective amount" of the disclosed compound of the present invention is the quantity that
would result in greater control of blood glucose concentration than in the absence of
treatment. An "effective amount" of a compound of the present invention administered
to a subject may depend on the type and severity of the disease and on the characteristics
of the subject including, without limitation, general health, age, sex, body weight,
tolerance to drugs, and the severity of inability to regulate blood glucose.
As used herein, the term "treating" is understood to mean amelioration of the
symptoms associated with a specific disorder or condition, such as hypoglycemia.
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.
As used herein, "fibrillation" refers to gelation and soluble aggregate formation
observed when glucagon is formulated at low or high pH.
Example 1: Peptide Synthesis
The compound of SEQ ID NO: 2 is generated by solid-phase peptide synthesis on
a Protein Technologies Inc. Symphony. Synthesis (0.125 mmols scale) is performed on
Fmoc- Glu(OtBu)-Wang polystyrene resin (Advanced ChemTech) with substitution
approximately 0.9 mmol/g. The synthesis is performed using the Fmoc main-chain
protecting group strategy. Amino acid side-chain derivatives used are: Asp(0-tert-butyl,
OtBu), Gln(Trityl, Trt), Glu(OtBu), His(Trt), Lys(tert-butoxy-carbonyl, Boc), Ser(OtBu),
Thr(OtBu), Trp(Boc), and Tyr(OtBu). Coupling is carried out with approximately 10
equivalents of amino acid activated with diisopropylcarbodiimide (DIC) and
hydroxybenzotriazole (HOBt) (1:1:1 molar ratio) in dimethylformamide (DMF).
Coupling is carried out for 90 minutes to 4 hours at room temperature.
Concomitant cleavage from the resin and side chain protecting group removal is
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 room temperature. The
solution is filtered and peptide is precipitated with cold diethyl ether and centrifuged at
4000 rpm for 3 min (cold ether washing repeated for three times). Crude peptide is
redissolved in 100-150 mL of water containing 10% acetic acid and purified on a C
reversed-phase high performance liquid chromatography (HPLC) column (typically a
Waters SymmetryPrep 7 um, 19 x 300 mm) at a flow rate of 18 mL/min. Sample is eluted
with a linear AB gradient of 22 to 55 % B over 100 minutes where A = 0.05% TFA/water
and B = 0.04 % TFA/acetonitrile. Product generally elutes at about 32-35 % acetonitrile.
Peptide purity and molecular weight is confirmed on an Agilent 1100 Series liquid
chromatography-mass spectrometry (LC-MS) system with a single quadrupole MS
detector. Analytical HPLC separation is done on a Waters SymmetryShield RP18, 3.5
micron, 4.6 mm x 100 mm column with a linear AB gradient of 10 to 100 % B over 15
minutes in which A = 0.05 % TFA/H20 and B = 0.04 % TFA/ 40% water/ 60%
acetonitrile and the flow rate is 0.7 mL/min (wavelength of 220 hih) . The peptide
analogue is purified to > 95 % purity and is confirmed to have molecular weight
corresponding to the calculated value within 1 atomic mass unit (amu).
The TFA salt is converted to the acetate salt using AG 1-X8 Resin (Bio-RAD,
acetate form, 100-200 mesh, 3.2 meq/dry g, moisture content 39-48% by wt) (anion
exchange resin). For example, 464 mg peptide is dissolved in 120 mL of 30%
Acetonitrile/H 20 . 30 g resin (about 100 fold molar ratio to positive charges of the
peptide) is added. The mixture solution is mixed by rotary stirring at room temperature
for 1 hour. The mixture solution is filtered, and the resin is washed 5 times with 30%
ACN/H20 . The original solution and washed solution are combined and lyophilized.
Solubility and Chemical stability
The compound of SEQ ID NO: 2 is dissolved in 20 mM Tris-HCl/H20 , pH 8 to a
1 mg/mL concentration (peptide content), and filtered through a 0.22 micron filter
(Millex, SLGV004SL). Solution is transferred to three vials: one for 4°C; one for 30°C
and one for 40°C. All vials are autoclaved. Samples are visually assessed at different time
points for turbidity and phase separation. Stability of the compound is assessed by
analytical HPLC on a Phenomenex Aeris Widepore, 3.6 mih, XB-C18 4.6 x 250 mm
column (P/NO 00G-4482-E0) heated at 60°C with a AB (A=0.05 %TFA/H20 ; B= 0.04
%TFA/acetonitrile ) gradient of 5 % B isocratic over 5 min, 5 to 30 % B over 20
minutes, 30 to 35 % B over 30 min, and 35 % to 45 % B over 10 min with a flow rate of
1.2 mL/min (wavelength of 220 hih) .
The compound of SEQ ID NO: 2 of the present invention maintains good solubility at
4°C, 30°C and 40°C, in Tris-HCl buffer at pH8 in 4-wk both by visual assessment and by
RP-HPLC.
TABLE 1
The compound of SEQ ID NO: 2 maintains chemical stability at 4°C, 30°C and 40°C, at
pH 8 in 4-wk both by visual assessment and by RP-HPLC. RP-HPLC main peak
changes: < 2.5% (4-wk 30°C vs 4°C); <7% (4-wk 40°C vs 4°C) (see Table 2).
TABLE 2
Physical stability test using Thioflavin T binding assay
Fibrillation is a common problem when glucagon is formulated in aqueous
solution. To assess the level of fibrillation of the compound of the present invention, a
Thioflavin T binding assay is performed.
The compound of SEQ ID NO: 2 is dissolved in various test buffers at 1 mg/mL
in 2.5 mL Fisher vials with a flat bottom (Fisher FS60965D) containing a flea sized
stirring bar (Fishers Catalog # 1451364). Test buffers are prepared in H20 and are all
adjusted to pH 8.0:
Buffer 1= 20 mM Tris-HCl
Buffer 2= 20 mM Tris-HCl, 150 nM NaCl
Buffer 3= 20 mM Tris-HCl, 300 mM sucrose
Buffer 4= 20 mM Tris-HCl, 300 mM sorbitol
Buffer 5= 20 mM Tris-HCl, 0.02% Tween 80
In addition, human glucagon (SEQ ID NO: 1) is dissolved in a 12 mg/mL glycerol
solution at pH 2.8 to a final concentration of 1 mg/mL. All samples are mechanically
stressed at 25 °C in a magnetic stir plate set at 300 rpm. Aliquots of the different samples
(100 m each aliquot and done in triplicates) are taken at time points 0, 40 and 120 hours,
and are added to a plate followed by 10 of a 1 mM Thioflavin T (stock solution in
H20 , pH 2.8)( T35516-25G, Sigma Aldrich). Samples are incubated for 30 min.
Fluorescence is measured using a Spectramax M5 (Moleculer Devices) using 440 hih as
the excitation wavelength, and the emission wavelength is set at 480 hih with a 475 hih
cut off and automatic sensitivity adjustment. Raw data is collected with Softmax Pro
5.4.1 (Molecular Devices) and imported to Excel. The average of the 3 wells per each
time point becomes the reported flurescence units shown in Table 3 below:
TABLE 3
Sample t = 0 h t = 40 h t = 120 h
193.9 201.6 226.1
SEQ ID NO: 2 in Buffer 1
182.2 210.1 213.6
SEQ ID NO: 2 in Buffer 2
263.3 297.0 310.9
SEQ ID NO: 2 in Buffer 3
260.9 285.1 311.2
SEQ ID NO: 2 in Buffer 4
251.7 281.7 306.8
SEQ ID NO: 2 in Buffer 5
Human glucagon (SEQ ID NO: 1) in 12 mg/mL 37.1 462.7 737.1
glycerol, pH 2.8
32.4 32.5 36.5
Buffer 1
33.6 44.2 34.6
Buffer 2
46.4 47.4 50.5
Buffer 3
38.8 38.4 38.2
Buffer 4
33.1 35.2 35.0
Buffer 5
33.3 36.7 37.4
12 mg/mL glycerol, pH 2.8
As shown in Table 3, the compound of SEQ ID NO: 2 maintains physical stability at
25°C and pH 8 for 120 hours in the presence of mechanical stress as assessed by both
visual assessment and Thioflavin T binding assays. The compound of SEQ ID NO: 2 did
not demonstrate significant fibrillation as measured by the Thioflavin T binding assay.
Effects of compound on blood glucose levels in C57/B16 male mice
To determine the effects of the compound of SEQ ID NO: 2 on blood glucose
levels, the compound is administered to C57/B16 mice. Three month old male C57BL6
mice (Harlan Laboratories) are used. Animals are individually housed in a temperaturecontrolled
(24°C) facility with a 12 hour light/dark cycle, and have free access to food
and water. After 1 week acclimation to the facility, mice are randomized to treatment
groups (n= 4/group). Test compound is formulated in Buffer 2 (see Physical stability test
using Thioflavin T binding assay). On the morning of test, food is removed at 08:00 AM.
Two hours after food is removed, the test compound is given subcutaneously at 0, 1, 3 or
10 g/kg doses. Blood glucose is measured at time 0, 15, 30, 60 and 120 minutes after
test compound administration with an ACCU-CHECK ® (Roche Diagnostics) glucometer.
Table 4 shows the glucose values at different time points. Results are expressed as mean
+ standard error mean (SEM) of 4 mice per group.
ED5 0 is calculated on the 30 minute glucose measurements. Blood glucose levels
at 10 g/kg of compound of SEQ ID NO: 2 is taken as the maximum value. For the
compound of SEQ ID NO: 2, the ED50 is 3.34 g/kg (95% confidence interval). The
results demonstrate that the compound of SEQ ID NO: 2 is able to increase blood
glucose.
TABLE 4
Human Glucagon Receptor Binding Assay
The binding of the compound of SEQ ID NO: 2 is determined by using a
293HEK cell line overexpressing the human glucagon receptor (hGR) (Lok S et al. Gene
140 (2), 203-209 (1994); GenBank: L20316).
Crude plasma membranes are prepared using cells from suspension or adherent
culture. The cell pellets are lysed on ice in a hypotonic homogenization buffer (25 mM
Tris HC1, pH 7.5, 1mM MgCl2, and Roche Complete™ Inhibitors without EDTA
(Roche, 11873580001)) with DNAase at 20 g/ml (Invitrogen, 18047-019). The cell
suspension is homogenized with a glass dounce homogenizer using a Teflon pestle for 25
strokes. The homogenate is centrifuged 1800 X g at 4 °C for 15 min. The supernatant is
collected and the pellet is resuspended in hypotonic homgenization buffer (without
DNAse) and re-homogenized. The mixture is centrifuged at 1800 X g for 15 min. The
second supernatant is combined with the first supernatant and centrifuged at 1800 X g for
15 min to clarify. This clarified supernatant is further centrifuged at 25000 X g for 30
min at 4 °C. The membrane pellet is resuspended in hypotonic homogenization buffer
(without DNAse) and stored as frozen aliquots at -80 °C until use.
Human glucagon is radioiodinated by 5I-lactoperoxidase procedure and purified
by reversed phase HPLC at Perkin-Elmer/NEN (NEX207). The specific activity is about
2200 Ci/mmol. ¾ determination is performed by homologous competition instead of
saturation binding due to high propanol content in the 1 5I-labelled glucagon material.
The K is estimated to be 1.24 hM and is used to calculate Ki values for all compounds
tested.
The receptor binding assay is carried out using a Scintillation Proximity Assay
(SPA) (Sun, S., Almaden, J., Carlson, T.J., Barker, J . and Gehring, M.R. Assay
development and data analysis of receptor- ligand binding based on scintillation proximity
assay. Metab Eng. 7 :38-44 (2005)) with wheat germ agglutinin (WGA) beads (Perkin-
Elmer) previously blocked with 1% fatty acid free bovine serum albumin (BSA) (Gibco,
7.5% BSA) Human glucagon (SEQ ID NO: 1) and compound (SEQ ID NO: 2) are
dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mM and stored frozen at
-20 °C.
Human glucagon and compound of SEQ ID NO: 2 are serially diluted into
DMSO. 10 E of diluted samples are transferred into Corning 3632 clear bottom assay
plates containing 40 E assay Binding Buffer (25 mM 4-(2-hydroxyethyl)-lpiperazineethanesulfonic
acid (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)
or cold glucagon (non-specific binding (NSB) at 1 mM final). 90 E membranes (3
mg/well), 50 mE 5I-labelled Glucagon (0.15 hM final concentration in reaction), and 50
mE of WGA beads (150 mg/well) are added. DMSO concentration does not exceed 4.2%.
Plates are sealed, mixed end over end, and read with a MicroBeta® scintillation counter
after 12 hours of settling time at room temperature.
125 Results are calculated as a percent of specific I-labelled glucagon binding in the
presence of compound. The absolute IC50 concentration of compound is derived by nonlinear
regression of percent specific binding of 1 5I-labelled glucagon vs. the
concentration of sample added (8.5xl0 ~12 to 0.5xl0 7 mol/L). The IC50 dose is converted
to Ki using the Cheng-Prusoff equation (Cheng Y., Prusoff W. H., Biochem. Pharmacol.
22: 3099-3108 (1973)). The Ki of the compound of SEQ ID NO: 2 was 3.20 + 0.76 hM
(n=3) for hGR binding (Ki for human glucagon was 1.66 + 0.09 hM (n=47) for hGR
binding). This data demonstrates that the compound of SEQ ID NO: 2 binds to hGR with
similar affinity compared to human glucagon and may activate that receptor, in turn
triggering glucagon-dependent physiological responses.
Mouse Glucagon Receptor Binding Assay
To determine whether the compound of SEQ ID NO: 2 binds to the mouse
glucagon receptor (mGR), a binding assay as essentially described in the Human
Glucagon Receptor Binding Assay is performed. Crude plasma membranes are prepared
from 293HEK cells in suspension culture expressing a cloned mGR. ((Burcelin R, Li J,
Charron MJ. Gene 164 (2), 305-10 (1995) GenBank: L38613). Membrane pellets are
prepared as described in the Human Glucagon Receptor Binding Assay, resuspended in
homogenization buffer and stored as frozen aliquots at -80 °C until use.
Human glucagon is radioiodinated by 125I-lactoperoxidase procedure and purified
by reversed phase HPLC at Perkin-Elmer/NEN (NEX207). The specific activity is about
2200 Ci/mmol. K determination is performed by homologous competition instead of
saturation binding due to high propanol content in the 125I-labelled glucagon material. The
K is estimated to be 2.05 hM and is used to calculate Ki values for all compounds tested.
The SPA receptor binding assay and calculation of the results are carried out as
described in the Human Glucagon Receptor Binding Assay. The Ki of the compound of
SEQ ID NO: 2 was 8.63 + 0.71 hM (n=3) for mGR binding (Ki for human glucagon was
1.37 + 0.07 hM (n=33) for mGR binding). This data demonstrates that compound of
SEQ ID NO: 2 binds to mGR and may activate that receptor, in turn triggering glucagondependent
physiological responses.
Glucagon-Like-Peptide 1 Receptor Binding Assay
To determine whether the compound of SEQ ID NO: 2 binds to the human
glucagon-like peptide 1 receptor (hGLP-lR), a binding assay as essentially described in
the Human Glucagon Receptor Binding Assay is performed. Crude plasma membranes
are prepared from 293HEK suspension cells expressing a 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) GenBank: NM_002062)
isolated from 293HEK membranes. Membrane pellets are prepared as described in the
Human Glucagon Receptor Binding Assay, resuspended in homogenization buffer and
stored as frozen aliquots at -80 °C until use.
Glucagon-like peptide 1 7-36 amide (GLP-1 amide) (SEQ ID NO: 3) is
radioiodinated by the 125I-lactoperoxidase procedure and purified by reversed phase
HPLC at Perkin-Elmer/NEN (NEX308). The specific activity is about 2200 Ci/mmol.
K determination is performed by homologous competition instead of saturation binding
1 5 due to high propanol content in the I-labelled GLP- 1 amide material. The K is
estimated to be 0.329 hM and is used to calculate Ki values for all compounds tested.
The SPA receptor binding assay and calculation of the results are carried out as
described in the Human Glucagon Receptor Binding Assay with the exception that
radioiodinated GLP-1 amide is used instead of the radioiodinated glucagon of the Human
Glucagon Receptor Binding Assay.
The Ki of the compound of SEQ ID NO: 2 was >3380 hM (n=3) for hGLP-lR
binding while the Ki of glucagon (SEQ ID NO: 1) was 2098 + 9 1 (n=17) (Ki for human
GLP-1 7-36 amide was 0.427 + 0.169 hM (n=64) for hGLP-lR binding). This data
demonstrates that the compound of SEQ ID NO: 2 does not specifically bind to hGLP-lR
and thus does not initiate GLP-1R-mediated physiological responses.
Glucose-Dependent Insulinotropic Peptide Receptor Binding Assay
To determine whether the compound of SEQ ID NO: 2 binds to the glucosedependent
insulinotropic peptide receptor (GIP-R), a binding assay as essentially
described in the Human Glucagon Receptor Binding Assay is performed. Crude plasma
membranes are prepared from suspension Chinese Hamster Ovary cells (CHO-S)
expressing human GIP-R (R (Usdin,T.B., Gruber,C, Modi,W. and Bonner,T.I., GenBank:
AAA84418.1) using cells from suspension culture. Membrane pellets are prepared as
described in the Human Glucagon Receptor Binding Assay, resuspended in
homogenization buffer and stored as frozen aliquots at -80 °C until use.
GIP (SEQ ID NO: 4) is radioiodinated by the I-125-lactoperoxidase procedure
(Markalonis, J.J., Biochem. J. 113:299 (1969)) and purified by reversed phase HPLC at
Perkin-Elmer/NEN (NEX-402). The specific activity is 2200 Ci/mmol. KD
determination is performed by homologous competition using cold human GIP instead of
saturation binding. The K is estimated to be 0.174 hM and is used to calculate Ki values
for all compounds tested.
The SPA receptor binding assay and calculation of the results are carried out as
described in the Human Glucagon Receptor Binding Assay The Ki of the compound of
SEQ NO: 2 was >2240 hM (n=l) for human GIP-R binding while the Ki of glucagon
(SEQ ID NO: 1) was >3010 (n=l) (Ki for human GIP was 0.127 + 0.048 hM) . This data
demonstrates that the compound of SEQ ID NO: 2 does not specifically bind to hGIP-R
and thus does not initiate hGIP-R-mediated physiological responses.
Human Glucagon Receptor Stimulated cAMP Functional Assay.
The hGR stimulated cAMP functional assay uses the same cloned hGR expressing
cell line as used for the hGR binding assay described above in the Human Glucagon
Receptor Binding Assay Cells are stimulated with glucagon, buffer controls, or Test
samples, and the cAMP generated within the cell is quantitated using the CisBio cAMP
Dynamic 2 HTRF Assay Kit (62AM4PEC). Briefly, cAMP levels within the cell are
detected by binding to the cAMP-d2 capture antibody in the presence of cell lysis buffer.
A second detection antibody provided in the kit, anti-cAMP Cryptate, is added to create a
competitive sandwich assay. When the detection antibody complex formed there is an
increase in the signal that is measured on a Perkin-Elmer Envision® instrument.
The hGR-HEK293 cells are harvested from sub-confluent tissue culture dishes
with Enzyme-Free Cell Dissociation Solution (Specialty Media 5-004-B). The cells are
pelleted at 100 X g at room temperature for 5 minutes then washed twice with phosphate
buffered saline (PBS). The washed cell pellet is resuspended at l x 107 cells/ml in
Recovery™ Freeze Media (Gibco 2044) and frozen in liquid nitrogen. On the day of
treatment, a frozen aliquot of cells is transferred into pre-warmed Resuspension Cell
Media (DMEM, Gibco (31053P) containing 0.5% defined FBS (Hyclone SH30070); 20
mM HEPES, pH 7.4; and 2 mM Glutamine). The cells are then pelleted at 100 X g at
room temperature for 5 minutes. The supernatant is removed and the cell pellet is
resuspended in Cell Media (DMEM, Gibco (3 1053P) with 0.1% fatty acid-free bovine
serum albumin, BSA, 7.5%, (Gibco 15620); 20 mM HEPES, pH 7.4, and 2 mM
Glutamine) at 1.25xl0 5 cells/ml. Test samples are prepared as 2 mM stocks in DMSO
and frozen at -20°C until needed. Glucagon, buffer controls and compound of SEQ ID
NO: 2, are serially diluted into DMSO followed by a step-down dilution into Compound
Dilution Media (Assay Media (DMEM, Gibco 31053P with 0.1% fatty acid-free bovine
serum albumin, BSA, 7.5%, (Gibco 15620); 20 mM HEPES, pH 7.4, and 2 mM
Glutamine) that contains 500 mM IBMX). The reaction is performed in 40 E, by adding
20 of cells (2500 cell/well) or cAMP standard curve samples to 96 Well plate Half
Area Black plates (Costar 3694), followed by addition of 20 m of either 2X
concentrated glucagon, buffer controls or compound of SEQ ID NO: 2 in Compound
Dilution Media. Final DMSO concentration does not exceed 1.1%, and final IBMX
concentration is 250 mM. The reaction is stopped by addition of 20 E of the cAMP-d2-
capture antibody (CisBio) diluted into the CisBio lysis buffer then gently mixed in
TITERTEK shaker. After 5 minutes of lysis, 20 mE of the detection antibody, anti-cAMP
Cryptate (CisBio), is added and mixed at 600 rpm for 1 minute. The lysed cell and
antibody mixtures are read after 1 hour at room temperature using the Perkin-Elmer
Envision®. Envision® units were converted to pmol/L cAMP/well using the cAMP
standard curve. The picomoles of cAMP generated in each well is converted to a percent
of the maximal response observed with the glucagon control. A relative EC5 0 value is
derived by non-linear regression analysis using the percent maximal response vs. the
concentration (0.17xl0 12 to lxlO 8 M) of peptide added.
The compound of SEQ ID NO: 2 bound hGR with an EC50 of 0.0658 ±0.0167 hM
(n=8) (EC 50 for human glucagon was 0.0142+0.0018 hM (n=6)). This data demonstrates
that the compound of SEQ ID NO: 2 binds and activates hGR and can thereby initiate
glucagon receptor-mediated physiological responses.
Sequence Listing
Human glucagon:
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-
Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ
ID NO: 1)
Example 1 :
Tyr-Ser-His-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Lys-Tyr-Leu-Asp-
(Aib) -Lys-Lys-Ala-Ala-Glu-Phe-Val-Ala-Trp-Leu-Leu-Glu-Glu
(SEQ ID NO: 2)
Human GLP-1:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-
Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-
NH2 (SEQ ID NO: 3)
Human GIP:
Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-
Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-
Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln (SEQ ID NO:4)

WE CLAIM:
1. A compound comprising the amino acid sequence of Tyr-Ser-His-Gly-Thr-Phe-
Thr-Ser-Asp-Val-Ser-Lys-Tyr-Leu-Asp-(Aib)-Lys-Lys-Ala-Ala-Glu-Phe-Val-
Ala-Trp-Leu-Leu-Glu-Glu (SEQ ID NO: 2).
2. The compound of Claim 1, wherein the compound consists of the amino acid
sequence of Tyr-Ser-His-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Lys-Tyr-Leu-Asp-
(Aib)-Lys-Lys-Ala-Ala-Glu-Phe-Val-Ala-Trp-Leu-Leu-Glu-Glu (SEQ ID NO: 2).
3. A pharmaceutical composition comprising the compound of Claim 1 or Claim 2
and a pharmaceutically acceptable carrier.
4. A method of treating hypoglycemia in a subject comprising administering an
effective amount of a compound of Claim 1 or Claim 2.
5. A compound of Claim 1 or Claim 2 for use in a therapy.
6. A compound of Claim 1 or Claim 2 for use in the treatment of hypoglycemia.
7. A compound of Claim 1 or Claim 2 for use in the manufacture of a medicament
for the treatment of hypoglycemia.