Methods for treating metabolic disorders and obesity with a peptide comprising the amino acid ...

188 Pages·2015·6.98 MB·English

US008975223B2
(12) United States Patent (10) Patent N0.: US 8,975,223 B2
\?gnatietaL (45) Date of Patent: b1ar.10,2015
(54) METHODS FOR TREATING METABOLIC (56) References Cited
DISORDERS AND OBESITY WITH A
PEPTIDE COMPRISING THE AMINO ACID U.S. PATENT DOCUMENTS
SEQUENCE OF SEQ ID NO. 146
2008/0286808 A1 11/2008 Schellenberger et al.
(75) Inventors: Louis Vignati, Indianapolis, IN (US);
FOREIGN PATENT DOCUMENTS
Richard D. DiMarchi, Carmel, IN (US)
W0 WO 2009/023270 2/2009
(73) Assignee: Marcadia Biotech, Inc., Nutley, NJ W0 WO 2009/099763 8/2009
(Us) W0 WO 2010/011439 1/2010
W0 WO 2010/071807 6/2010
Notice: Subject to any disclaimer, the term of this W0 WO 2010/148089 12/2010
W0 WO 2011/094337 8/2011
patent is extended or adjusted under 35
W0 WO 2012/088116 6/2012
U.S.C. 154(b) by 0 days.
OTHER PUBLICATIONS
(21) Appl. No.: 13/996,937
Perfetti et al.. Eur. J. Endocr. 143, 717-725, 2000.*
Tseng et al., PNAS, 90: 1992-1996, 1993*
(22) PCT Filed: Dec. 22, 2011
Adelhorst et al., J. Biol. Chem. 269: 6275-6278, 1994*
Charpentier, Oral combination therapy for type 2 diabetes, Diabetes
(86) PCT No.: PCT/US2011/066739 Metab. Res. Rev., 18 Suppl 3:S70-6 (2002).
Irwin et al., Antidiabetic effects of sub-chronic activation of the GIP
§ 371 (0X1)’
receptor alone and in combination with background exendin-4
(2), (4) Date: Sep. 20, 2013 therapy in high fat fed mice, Regul. Pept., 153(1-3):70-6 (2009).
Lteif et al., Insulin resistance, metabolic syndrome and vascular
(87) PCT Pub. No.: WO2012/088379 diseases: update on mechanistic linkages, Can J. Cardiol, 20 Suppl
B166B-76B (2004).
PCT Pub. Date: Jun. 28, 2012 Mathur, “Metabolic Syndrome,” downloaded from the Internet at:
<http://www.medicinenet.com/> (last editorial review Sep. 27,
(65) Prior Publication Data 2010).
Nauck et al., Additive insulinotropic effects of exogenous synthetic
US 2014/0018291A1 Jan. 16, 2014 human gastric inhibitory polypeptide and glucagon-like peptide-1
(7-36) amide infused at near-physiological insulinotropic hormone
and glucose concentrations, J. Clin. Endocrinol. Metab., 76(4):912-7
Related US. Application Data (1993).
Resnick et al., Achievement of American Diabetes Association clini
(60) Provisional application No. 61/500,229, ?led on Jun.
cal practice recommendations among US. adults with diabetes,
23, 2011, provisional application No. 61/426,338, 1999-2002: the National Health and Nutrition Examination Survey,
?led on Dec. 22, 2010. Diabetes Care, 29(3):531-7 (2006).
(51) Int. Cl. * cited by examiner
A61K 38/26 (2006.01)
A61P 3/10 (2006.01) Primary Examiner * Gyan Chandra
A61K 31/195 (2006.01) (74) Attorney, Agent, or Firm * Marshall, Gerstein & Borun
A61K 31/198 (2006.01) LLP
A61K 45/06 (2006.01)
(52) US. Cl. (57) ABSTRACT
CPC ............. .. A61K 38/26 (2013.01); A61K31/195 Methods are provided for administering an extended half-life
(2013.01); A61K31/198 (2013.01); A61K GLP-l/GIP coagonist peptide to a patient in need thereof for
45/06 (2013.01) reducing weight gain, inducing weight loss, treating hyperg
USPC .............. .. 514/68; 514/69; 514/7.2; 514/73 lycemia, reducing blood glucose levels, or normalizing blood
(58) Field of Classi?cation Search glucose levels in said patient.
None
See application ?le for complete search history. 11 Claims, 5 Drawing Sheets
US. Patent
Mar. 10, 2015 Sheet 1 0f 5 US 8,975,223 B2
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US. Patent Mar. 10, 2015 Sheet 2 0f5 US 8,975,223 B2
250
|:| Placebo
200 I Coagonist Peptide 4 mg
5 1
g 150
q:
8
o 100 '
E
(D
50
0 0 2 4 6 8 12
Glucose Infusion Rate mg/kg/min
FIG. 2A
US. Patent Mar. 10, 2015 Sheet 3 0f5 US 8,975,223 B2
250
200 [lPlacebQ I
:é I Coagonlst Pept|de 8mg
5 150
CD
§ 100 —
E
(D
50 -
0 - | | | | | | | | | | | |
0 2 4 6 8 12
Glucose Infusion Rate mg/kg/min
FIG. ZB
US. Patent Mar. 10, 2015 Sheet 4 0f5 US 8,975,223 B2
6.00
5.00 U 4mg
A I 8mg
E 4.00 I111] 16mg
0)
5
g 3.00
a
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0 2 4 0 8 12
Glucose Infusion Rate mg/kg/min
FIG. 3

US 8,975,223 B2
1 2
METHODS FOR TREATING METABOLIC thereof (e. g., the adult human has excess body weight, diabe
DISORDERS AND OBESITY WITH A tes mellitus type II, insulin resistance, alcoholic liver disease,
PEPTIDE COMPRISING THE AMINO ACID non-alcoholic fatty liver disease (NAFLD), or metabolic syn
SEQUENCE OF SEQ ID NO. 146 drome) by administering to the adult human an extended
half-life GLP-l/GIP coagonist peptide at a total weekly dos
PRIORITY CLAIM
age of about 1 mg to about 40 mg, or about 4 mg to about 30
mg, or about 4 to about 20 mg, or about 10 to about 20 mg, or
This application claims priority to International Applica
about 2 to about 10 mg.
tion No. PCT/US2011/066739, ?led Dec. 22, 2011, US.
Another aspect of the invention provides methods for treat
Provisional Application No. 61/500,229, ?led Jun. 23, 2011,
ing hyperglycemia, reducing blood glucose levels, or normal
and US. Provisional Application No. 61/426,338, ?led Dec.
iZing blood glucose levels in an adult human in need thereof
22, 2010; the contents of each application are incorporated
herein by reference. (e.g., the adult human has diabetes mellitus type I, diabetes
mellitus type II, or gestational diabetes) by administering to
INCORPORATION BY REFERENCE OF the adult human an extended half-life GLP-l/GIP coagonist
MATERIAL SUBMITTED ELECTRONICALLY peptide at a total weekly dosage of about 1 mg to about 40 mg,
or about 4 mg to about 30 mg, or about 4 to about 20 mg, or
Incorporated by reference in its entirety is a computer about 10 to about 20 mg, or about 2 to about 10 mg.
readable nucleotide/amino acid sequence listing submitted The extended half-life GLP-l/GIP coagonist peptide typi
concurrently herewith and identi?ed as follows: One 285,688 cally has a GIP percentage potency of at least 1%, or a GLP-l
byte ASCII (Text) ?le named “45708A_SeqListing.txt,” cre 20 percentage potency of at least 1%. In some embodiments, the
ated on Dec. 22, 2011. extended half-life GLP-l/GIP coagonist peptide exhibits a
GLP-l percentage potency within about 10-fold of the GIP
BACKGROUND OF THE INVENTION
percentage potency. Alternatively, in some embodiments, the
extended half-life GLP-l/GIP coagonist peptide exhibits an
1. Field of the Disclosure
25 EC50 at the GLP-1 receptor within about 10-fold of the EC50
This disclosure relates generally to methods of administer
at the GIP receptor. In any of these embodiments, for
ing an extended half-life GLP-l/GIP coagonist peptide. More
example, the extended half-life GLP- 1 /GIP coagonist peptide
particularly, the disclosure relates to methods for reducing
weight gain or inducing weight loss, and methods for treating exhibits a half-life of about 4 to about 10 days, or about 4 to
hyperglycemia, reducing blood glucose levels, or normaliZ about 7 days. In any of these embodiments, the administration
ing blood glucose levels through the administration of an 30 of the extended half-life GLP-l/GIP coagonist peptide can
extended half-life GLP-l/GIP coagonist peptide. result in an increase in insulin level, a decrease in glucose
2. Brief Description of Related Technology level, an increase in C-peptide level, a decrease in HbAlc
Diabetes mellitus type II (i.e., type 2 diabetes) is a hetero level, or a decrease in fructosamine level, or any combination
geneous group of conditions that constitute approximately thereof.
90% of diabetes in the United States. Type 2 diabetes is 35 The extended half-life GLP-l/GIP coagonist peptide can
caused by a combination of insulin resistance and diminished be administered alone or in combination with a second thera
insulin secretion. Weight reduction in obese patients is asso peutic agent (e.g., an anti-diabetic agent and/or an anti-obe
ciated with improvement of insulin resistance and ameliora sity agent), and at any frequency that results in the recited
tion of diabetes symptoms. total weekly dose (e.g., administered once per week, twice
There are ?ve widely recognized classes of oral anti-dia 40 per week).
betic agents, sulfonylureas, biguanides, meglitinides, thiazo In some embodiments, the extended half-life GLP-l/GIP
lidinediones, and alpha-glucosidase inhibitors. Treatment of coagonist peptide includes one or more hydrophilic polymer
type 2 diabetes usually involves choosing one or more of moieties that have a collective molecular weight of about
these oral agents as initial therapy (see, e.g., Charpentier G. 30,000 Daltons to about 60,000 Daltons (e.g., about 40,000
Diabetes Metab. Res. Rev. 18 (Supp. 3):570-576 (2002)). 45 Daltons). Nonlimiting examples of the extended half-life
Despite the use of multiple drugs, however, the all-over con GLP-l/GIP coagonist peptide include any of SEQ ID NOs:
trol of diabetes remains inadequate. Only 49.8% of persons 5-94, 99-169, 173-413, an amino acid sequence that has up to
with diabetes achieve the National Diabetes Association tar 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modi?cations relative
get HbAlc of less than 7%. Instead, 29.7% of persons with to SEQ ID NOs: 5-94, 99-169, 173-413 that retains, e.g., at
diabetes have an HbAlc of greater than 8% (see, e. g., Resnick 50 least 10% of the GIP or GLP-l activity, or a pegylated deriva
et al., Diabetes Care 29:531-537 (2006)). tive ofSEQ ID NOs: 5-94, 99-169, 173-413. Forexample, the
The incretin hormones, glucagon-like peptide-1 (GLP-1) extended half-life GLP-l/GIP coagonist peptide can include
and glucose dependent insulinotropic peptide (GIP), are natu any of SEQ ID NOs:75,99-103, 140, 153, 166, and 261.
rally-occurring peptide hormones. Both GLP-1 and GIP The extended half-life GLP-l/GIP coagonist peptide can
stimulate insulin synthesis and secretion in a glucose-depen 55 be administered in a sterile pharmaceutical composition, such
dent manner and do not produce hypoglycemia (see, e.g., as through subcutaneous, intravenous or intramuscular injec
Nauck et al., J. Clin. Endocrinol. Metab. 76:912-917 (1993) tion.
and Irwin et al., Regul. Pept. 153:70-76 (2009)).
GLP-l has been shown to be effective as adjunctive therapy BRIEF DESCRIPTION OF THE DRAWINGS
for diabetes. Although GLP-l therapy is associated with 60
weight loss, it is also associated with nausea, which occurs in FIG. 1 depicts the results of a Phase I, randomized, pla
over 20% of patients that are treated with GLP-l analogs. cebo-controlled, positive-controlled two-part study per
formed in healthy male and female subjects to evaluate the
SUMMARY effect of a range of doses on beta cell response to a glucose
65 load. Human subjects were administered placebo or 4 mg, 8
One aspect of the invention provides methods for reducing mg, or 16 mg of extended half-life GLP-l/GIP coagonist
weight gain or inducing weight loss in an adult human in need peptide and insulin secretion rates were measured. A dose
US 8,975,223 B2
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dependent, dose proportional, increase in insulin secretion De?nitions
rate (ISR) was observed after treatment with the extended As used herein, the term “peptide” encompasses a
half-life GLP-1/GIP coagonist peptide in comparison to pla sequence of 3 or more amino acids and typically less than
cebo. 100, or less than 50 amino acids, wherein the amino acids are
FIG. 2 depicts the results of a Phase I, randomized, pla naturally occurring or non-naturally occurring amino acids.
cebo-controlled, positive-controlled two-part study per Non-naturally occurring amino acids refer to amino acids that
formed in healthy male and female subjects to evaluate the do not naturally occur in vivo but which, nevertheless, can be
effect of a range of doses on beta cell response to a glucose incorporated into the peptide structures described herein.
load. Human subjects were administered placebo or 4 mg As used herein, the term “GLP-1/GIP coagonist peptide”
(FIG. 2A) or 8 mg (FIG. 2B) of extended half-life GLP-1/GIP refers to a peptide that exhibits activity at both the GLP-1 and
coagonist peptide and a glucose infusion at a rate between 0 GIP receptors. Such a peptide may optionally have no detect
and 12 mg/kg/min and plasma glucose levels were measured. able or relatively low glucagon activity (e.g., at least 10-fold
FIG. 3 illustrates the results of an experiment comparing lower percentage potency at the glucagon receptor compared
C-peptide levels in subjects administered 4 mg, 8 mg, or 16 to the GLP-1 or GIP receptors, or at least 100-fold lower
mg of extended half-life GLP-1/GIP coagonist peptide and a potency).
glucose infusion between 0 and 12 mg/kg/min. As used herein, the term “extended half-life” refers to a
FIG. 4 compares the appearance of plasma acetaminophen peptide having a prolonged duration of action and/or a
over a 4 hour time period in subjects administered BYETTA, decreased clearance rate. For example, a prolonged duration
or 4 mg, 8 mg, or 16 mg of extended half-life GLP-1/GIP 20 of action can be due to resistance to dipeptidase IV cleavage
coagonist peptide. and/or conjugation to a heterologous moiety that prolongs
half-life in circulation (e.g., polyethylene glycol (PEG), albu
DETAILED DESCRIPTION min, or an Fc region or fragment thereof).
As used herein the general term “polyethylene glycol” or
Extended half-life GLP-1/GIP coagonist peptides, as 25 “PEG,” refers to mixtures of condensation polymers of eth
described in PCT Patent Application Publication No. WO ylene oxide and water, in a branched or straight chain, repre
2010/01 1439, incorporated herein by reference in its entirety, sented by the general formula H(OCH2CH2)nOH, wherein n
were found to be useful for treating hyperglycemia, including is at least 9.
diabetes, as well as for reducing weight gain or inducing As used herein the term “pegylated” and like terms refers to
weight loss. These peptides provide greater weight reduction 30 a compound that has been modi?ed from its native state by
effects compared to GLP-1 alone. The extended half-life linking one or more polyethylene glycol moieties to the com
GLP-l/GIP coagonist peptides have a prolonged duration of pound. A “pegylated extended half-life GLP-l/GIP coagonist
action, e.g. due to a prolonged half-life in circulation (e.g., peptide” is an extended half-life GLP-1/GIP coagonist pep
about 5 to 6 days), which allows for dosing at longer intervals tide that has one or more PEG chains covalently bound to the
(e. g., weekly dosing, or even biweekly dosing). 35 extended half-life GLP-1/GIP coagonist peptide, e.g. at a
Without intending to be bound by any particular theory, the collective molecular weight of PEG of about 30,000 Daltons
extended half-life GLP-1/GIP coagonist peptides demon to about 60,000 Daltons.
strate similar or superior glucose-lowering and weight-reduc As used herein an amino acid “modi?cation” refers to a
tion effects compared to current GLP-1 therapies, but with a substitution, addition or deletion of an amino acid, and
reduced incidence of gastrointestinal tolerability problems 40 includes substitution with or addition of any of the 20 amino
such as nausea or vomiting. acids commonly found in human proteins, as well as atypical
The extended half-life GLP-1 GIP coagonist peptides have or non-naturally occurring amino acids. Throughout the
exhibited glucose normalization and weight lowering prop application, all references to a particular amino acid position
erties in a variety of animal models. In lean mice and rats, by number (e.g. position 28) refer to the amino acid at that
such peptides provide improved tolerance to a glucose chal 45 position in native glucagon (SEQ ID N011) or the corre
lenge. In diet-induced obese mice, such peptides reduced sponding amino acid position in any analogs thereof. For
both food intake and body weight gain. example, a reference herein to “position 28” would mean the
In Phase I, randomized, placebo-controlled, sequential, corresponding position 27 for a peptide in which the ?rst
single-ascending dose studies of the extended half-life GLP amino acid of SEQ ID NO: 1 has been deleted. Similarly, a
1/GIP coagonist peptide (see Example 1), single doses of the 50 reference herein to “position 28” would mean the correspond
extended half-life GLP-1/GIP coagonist peptide were admin ing position 29 for a peptide in which one amino acid has been
istered to healthy male volunteers, starting at 0.1 mg and added before the N-terminus of SEQ ID NO: 1. Commercial
ranging up to 32 mg, administered by subcutaneous (SC) sources of atypical amino acids include Sigma-Aldrich (Mil
injection. Each of the doses was well tolerated and not asso waukee, Wis.), ChemPep Inc. (Miami, Fla.), and Genzyme
ciated with adverse events, clinically important laboratory 55 Pharmaceuticals (Cambridge, Mass.). Atypical amino acids
?ndings, or ECG ?ndings. may be purchased from commercial suppliers, synthesized de
In a Phase I, randomized, placebo-controlled, positive novo, or chemically modi?ed or derivatized from other amino
controlled, two-part study (see Example 2), healthy male and acids.
female subjects in Part 1 of the study received a subcutaneous As used herein an amino acid “substitution” refers to the
(SC) injection of placebo and an SC injection of BYETTA® 60 replacement of one amino acid residue by a different amino
(Amylin Pharmaceuticals) as a positive control. Subjects in acid residue.
Part 2 of the study received an SC injection of placebo and an As used herein, the term “conservative amino acid substi
SC injection of the extended half-life GLP-1/GIP coagonist tution” is de?ned herein as the replacement of one amino acid
peptide. Doses of 4 mg, 8 mg, and 16 mg of the extended with another amino acid having similar properties, e. g., size,
half-life GLP-1/GIP coagonist peptide increased insulin 65 charge, hydrophobicity, hydrophilicity, and/or aromaticity,
secretion in a dose-dependent manner during a graded glu and includes exchanges within one of the following ?ve
cose infusion without an effect on gastric emptying. groups:
US 8,975,223 B2
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I. Small aliphatic, nonpolar or slightly polar residues: of the parent compound, and which are not biologically or
Ala, Ser, Thr, Pro, Gly; otherwise undesirable. Many of the compounds disclosed
II. Polar, negatively charged residues and their amides and herein are capable of forming acid and/or base salts by virtue
esters: of the presence of amino and/or carboxyl groups or groups
Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; similar thereto.
III. Polar, positively charged residues: Pharmaceutically acceptable base addition salts can be
His, Arg, Lys; Omithine (Om) prepared from inorganic and organic bases. Salts derived
IV. Large, aliphatic, nonpolar residues: from inorganic bases, include by way of example only,
Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine sodium, potassium, lithium, ammonium, calcium and mag
V. Large, aromatic residues: nesium salts. Salts derived from organic bases include, but are
Phe, Tyr, Trp, acetyl phenylalanine not limited to, salts of primary, secondary and tertiary amines.
As used herein the term “native glucagon” refers to a pep Pharmaceutically acceptable acid addition salts may be
tide consisting of the amino acid sequence of SEQ ID NO: 1, prepared from inorganic and organic acids. Salts derived from
the term “native GIP” refers to a peptide consisting of the inorganic acids include hydrochloric acid, hydrobromic acid,
amino acid sequence of SEQ ID NO: 4, and the term “native sulfuric acid, nitric acid, phosphoric acid, and the like. Salts
GLP-1” is a generic term that designates GLP-1(7-36) amide derived from organic acids include acetic acid, propionic
(consisting of the amino acid sequence of SEQ ID NO: 3), acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,
GLP-1(7-37) acid (consisting of the amino acid sequence of malonic acid, succinic acid, maleic acid, fumaric acid, tartaric
SEQ ID NO: 2) or a mixture of those two compounds.As used acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
herein, a general reference to “glucagon” or “GIP” or “GLP 20 methanesulfonic acid, ethanesulfonic acid, p-toluene-sul
1” in the absence of any further designation is intended to fonic acid, salicylic acid, and the like.
mean native glucagon or native GIP or native GLP- 1, respec GLP-l/GIP Coagonist Peptide Embodiments
tively. The invention described herein relates to methods of
As used herein a general reference to a peptide is intended administering an extended half-life GLP-l/GIP coagonist
to encompass peptides that have modi?ed amino and carboxy 25 peptide to an adult human in need thereof. GLP- 1/GIP coago
termini. For example, an amino acid chain comprising an nist peptides and extended half-life versions of such peptides
amide group in place of the terminal carboxylic acid is are exhaustively described in WO 2010/011439, hereby
intended to be encompassed by an amino acid sequence des incorporated by reference in its entirety.
ignating the standard amino acids. In some embodiments, the extended half-life GLP-l/GIP
As used herein, the term “selectivity” of a molecule for a 30 coagonist peptide is or comprises any of (a) SEQ ID NOs:
?rst receptor relative to a second receptor refers to the fol 5-94, 99-169, and 173-262, which correspond to SEQ ID
lowing ratio: EC50 of the molecule at the second receptor NOs: 5-94, 99-169, and 173-262 inWO 2010/01 1439, (b) any
divided by the EC50 of the molecule at the ?rst receptor. For of SEQ ID NOs: 263-277, which correspond to SEQ ID NOs
example, a molecule that has an EC50 of 1 nM at a ?rst 19-28 and 33-36 in US. Provisional Patent Application Ser.
receptor and an EC50 of 100 nM at a second receptor has 35 No. 61/ 29,8812, incorporated herein by reference, or (c) any
100-fold selectivity for the ?rst receptor relative to the second of SEQ ID NOs: 178-413, (d) any analogs of any of the
receptor. foregoing peptides having one or more amino acid modi?ca
As used herein, GLP-l percentage potency is the EC50 of tions, including up to 2, 3, 4, 5, 6, 7, 8, 9 or 10 further
GLP-1 divided by the EC50 of the extended half-life GLP-l/ modi?cations (including, e.g., conservative substitutions), or
GIP coagonist peptide, times 100%. 40 (e) pegylated derivatives of any of the foregoing peptides,
As used herein GIP percentage potency is the EC50 of GIP provided the analog retains the desired activity (e.g. at least
divided by the EC50 of the extended half-life GLP-l/GIP 1%, 10%, 20%, 30%, 40% or 50%) of the parent peptide at the
coagonist peptide, times 100%. GLP-1 and GIP receptors. WO 2010/011439 also provides
As used herein glucagon percentage potency is the EC50 of some guidance regarding amino acids which may be altered;
glucagon divided by the EC50 of the extended half-life GLP 45 in addition, the invention contemplates peptides with addi
1/GIP coagonist peptide, times 100%. tions, deletions, non-conservative substitutions and/or non
As used herein, the term “treating” includes prophylaxis of conservative substitutions at one or more of positions 1, 2, 5,
the speci?c disorder or condition, or alleviation of the symp 7, 8, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or29.
toms associated with a speci?c disorder or condition and/or The peptides and analogs may comprise additional heterolo
preventing or eliminating said symptoms. For example, as 50 gous moieties including one or more additional amino acid
used herein the term “treating diabetes” will refer in general sequences (e.g. as a fusion protein) or one or more PEG
to altering glucose blood levels in the direction of normal chains to result in pegylated derivatives of any of SEQ ID
levels and may include increasing or decreasing blood glu NOs: 1-413. In some exemplary embodiments, the extended
cose levels depending on a given situation. half-life GLP-l/GIP coagonist peptide is any of SEQ ID NOs:
The term, “parenteral” means not through the alimentary 55 75, 99-103, 140, 153, 166, and 261. It is understood that
canal but by some other route such as subcutaneous, intra analogs are prepared by de novo synthesis rather than by
muscular, intraspinal, or intravenous. preparing a parent peptide that is subsequently modi?ed,
As used herein, the term “pharmaceutically acceptable car although in some instances, e.g. pegylation, the parent pep
rier” includes any of the standard pharmaceutical carriers, tide may be prepared ?rst then pegylated.
such as a phosphate buffered saline solution, water, emulsions 60 In some embodiments, the EC50 of the extended half-life
such as an oil/water or water/oil emulsion, and various types GLP-l/GIP coagonist peptide at the GIP receptor is within
of wetting agents. The term also encompasses any of the about 20-fold, about 15-fold, or preferably within about
agents approved by a regulatory agency of the US Federal 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold,
government or listed in the US Pharmacopeia for use in or about 2-fold different (higher or lower) from its EC50 at the
animals, including humans. 65 GLP-1 receptor. In some embodiments, the extended half-life
As used herein the term “pharmaceutically acceptable salt” GLP-l/GIP coagonist peptide exhibits a GLP-l percentage
refers to salts of compounds that retain the biological activity potency within about 20-fold, about 15-fold, or preferably
US 8,975,223 B2
7 8
within about 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, acid (e.g., position 30) within a C-terminal extension (e.g.,
4-fold, 3-fold, or about 2-fold different (higher or lower) from 30, 31, 32, 33, 34, 35, 36, 37, 38, 39), or at the C-terminal
the GIP percentage potency. In exemplary embodiments (e.g., amino acid (e.g., 40, 41, 42, or higher), as described in WO
in any of the embodiments herein), the extended half-life 2010/011439. In exemplary embodiments, this hydrophilic
GLP-1/GIP coagonist peptide exhibits a GIP percentage polymer is covalently linked to a Lys, Cys, Orn, homocys
potency of at least about 1%, at least about 5%, or at least teine, or acetyl-phenylalanine residue at any of these posi
about 10%. In exemplary embodiments (e.g., in any of the tions. Exemplary hydrophilic polymers include one or more
embodiments herein), the extended half-life GLP-1/GIP polyethylene glycol (PEG) chains, for example, of a collec
coagonist peptide exhibits a GLP-1 percentage potency of at tive molecular weight of about or about 20,000 Daltons to
least about 1%. In some embodiments, the extended half-life about 40,000 Daltons, as described in WO 2010/011439. In
GLP-1/GIP coagonist peptide has about 10% or less of the some embodiments, the polyethylene glycol polymer has a
activity of native glucagon at the glucagon receptor, e.g. about molecular weight of about 30,000 Daltons to about 60,000
1-10%, or about 01-10%, or greater than about 0.1% but less Daltons, or about 30,000 Daltons to about 50,000 Daltons
than about 10%. In any of the embodiments herein, for (e.g., 40,000 Daltons).
example, the selectivity of the extended half-life GLP-1/GIP Heterologous Moieties
coagonist peptide for the GLP-1 receptor, compared to the As used herein, the term “heterologous moiety” is synony
GIP receptor, is less than 100-fold, or the ratio of the GLP-1 mous with the term “conjugate moiety” and refers to any
percentage potency divided by the GIP percentage potency is molecule (chemical or biochemical, naturally-occurring or
less than 100, less than about 20, 15, 10, 9, 8, 7, 6 or 5. non-coded) which is different from the peptide to which it is
The extended half-life GLP-1/GIP coagonist peptides have 20 attached. In some embodiments the heterologous moiety is
a prolonged duration of action, e. g. due to a prolonged half another peptide or protein. In some embodiments, the heter
life in circulation (e.g., about 5 to 6 days), which allows for ologous moiety is a plasma protein, e.g., albumin, transferrin,
dosing at longer intervals (e.g., weekly dosing, biweekly ?brinogen and globulins. In some embodiments the plasma
dosing). In some embodiments, the extended half-life GLP protein is albumin or transferrin.
1/GIP coagonist peptides used according to the invention 25 In some embodiments, the heterologous moiety is an Fc
have a half-life ranging from about 3 to about 15 days, e.g. portion, or fragment or modi?ed analog thereof, of an immu
about 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, noglobulins (Ig), e.g., IgG, IgA, IgE, IgD or IgM, preferably
3-15, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, a fragment that retains the FcRn binding site. The Fc region is
4-15, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-12, 5-14, 5-15, a C-terminal region of an Ig heavy chain, which is responsible
6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, or 6-15 days. In 30 for binding to Fc receptors (FcRn) that carry out activities
some embodiments, the extended half-life GLP-1/GIP such as recycling immunoglobulins and returning them to
coagoni st peptide undergoes complete clearance within about circulation (which results in prolonged half-life). For
10 days to about 30 days, for example about 20 days. example, according to some de?nitions the human IgG heavy
For example, a prolonged duration of action can be due to chain Fc region stretches from Cys226 to the C-terminus of
resistance or reduced susceptibility to dipeptidase IV cleav 35 the heavy chain. The region of the Fc portion of IgG that binds
age and/or conjugation to a heterologous moiety that pro to the FcRn receptor has been described based on X-ray
longs half-life in circulation. Resistance or reduced suscepti crystallography (Burmeister et al. 1994, Nature 3721379).
bility to dipeptidase IV cleavage is provided, e.g., by amino The major contact area of the Fc with the FcRn is near the
acid modi?cations (to a “DPP-IV protective amino acid”) at junction of the CH2 and CH3 domains and include amino acid
positions 1 and/or 2. Examples of suitable substitutions 40 residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and
include: at position 1, D-histidine, alpha, alpha-dimethyl imi 314 of the CH2 domain and amino acid residues 385-387,
diazole acetic acid (DMIA), N-methyl histidine, alpha-me 428, and 433-436 of the CH3 domain. Some conjugate moi
thyl histidine, imidazole acetic acid, desaminohistidine, eties may or may not include binding site(s) for FcyR, which
hydroxyl-histidine, acetyl-histidine or homo-histidine; or at are responsible for ADCC and CDC, or may include modi?
position 2, D-serine, D-alanine, valine, glycine, N-methyl 45 cations designed to reduce ADCC or CDC.
serine, or alpha-aminoisobutyric acid (AIB). In some embodiments, the heterologous moiety is a poly
Examples of heterologous moieties that prolong duration mer. In some embodiments, the polymer is selected from the
of action include, but are not limited to, acyl or alkyl groups; group consisting of: polyamides, polycarbonates, polyalky
dipeptide prodrug moieties (linked via, e.g., amide or ester lenes and derivatives thereof including, polyalkylene glycols,
bonds) or other prodrug moieties; polyethylene glycol (PEG) 50 polyalkylene oxides, polyalkylene terepthalates, polymers of
or other hydrophilic polymers; albumin or other plasma pro acrylic and methacrylic esters, including poly(methyl meth
teins; an Fc region of an immunoglobulin or a fragment acrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
thereof; rPEG. Such components are known in the art. For poly(isobutyl methacrylate), poly(hexylmethacrylate), poly
example, acylation or alkylation, hydrophilic polymers, albu (isodecyl methacrylate), poly(lauryl methacrylate), poly
min or other plasma proteins, and Fc regions of immunoglo 55 (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl
bulins or fragments thereof are described in Int’l Pub. No. WO acrylate), poly(isobutyl acrylate), and poly(octadecyl acry
2010/011439, hereby incorporated by reference in its late), polyvinyl polymers including polyvinyl alcohols, poly
entirety. Ester prodrug moieties are described in Int’l Pub. No. vinyl ethers, polyvinyl esters, polyvinyl halides, poly(vinyl
WO2009/099763, hereby incorporated by reference in its acetate), and polyvinylpyrrolidone, polyglycolides, polysi
entirety, and amide prodrug moieties are described in Int’l 60 loxanes, polyurethanes and co-polymers thereof, celluloses
Pub. No. WO 2010/071807, hereby incorporated by reference including alkyl cellulose, hydroxyalkyl celluloses, cellulose
in its entirety. Recombinant PEG, or rPEG, is described in ethers, cellulose esters, nitro celluloses, methyl cellulose,
Int’l Pub. No. WO2009/023270. ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl
In some embodiments, the extended half-life GLP-1/GIP methyl cellulose, hydroxybutyl methyl cellulose, cellulose
coagonist peptide comprises an amino acid that is covalently 65 acetate, cellulose propionate, cellulose acetate butyrate, cel
linked to a hydrophilic polymer at any of amino acid positions lulose acetate phthalate, carboxylethyl cellulose, cellulose
16, 17, 20, 21, 24, or 29, after position 29 at an added amino triacetate, and cellulose sulphate sodium salt, polypropylene,