LONG ACTING GLUCAGON LIKE POLYPEPTIDE-1 (GLP-1) RECEPTOR AGONISTS AND METHODS OF USE

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 1, 2022, is named 724939_IOT-056_SL.txt and is 489 kilobytes in size.

FIELD

The present invention relates to compounds that are glucagon like polypeptide-1 (GLP-1) receptor agonists and methods of preparing the same. The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders.

BACKGROUND

Endogenous GLP-1 is released from the gut in response to nutrient ingestion. Following food intake and digestion, carbohydrates and fats appear in the lumen of the gut, which stimulate a so-called incretin effect, the release of incretins such as GLP-1 from intestinal L-cells. GLP-1, once released, targets the pancreas where it enhances secretion of insulin in a “glucose dependent manner.” In other words, this GLP-1-mediated effect upon insulin persists when glucose levels are high yet safely dissipates as glucose levels fall. GLP-1 activity thus self-regulates to reduce the risk of hypoglycemia (the condition by which glucose levels drop dangerously low). Since GLP-1 has a short elimination half-life (t_1/2) of less than five minutes, this endogenous peptide is unsuitably short-lived for use as a therapeutic.

Synthetic analogs of GLP-1 have been designed to have longer half-lives and similarly enhance secretion of insulin in a glucose dependent manner like endogenous GLP-1, for use in the treatment of type 2 diabetes and for providing weight loss.

Certain GLP-1 receptor agonists, including Bydureon® (exenatide), marketed by AstraZeneca of Cambridge, U.K.; Trulicity® (dulaglutide), marketed by Eli Lilly and Co., of Indianapolis, Ind., U.S.A.; and Victoza® (liraglutide), Ozempic® (injectable semaglutide) & Rybelsus® (orally administered semaglutide), marketed by Novo Nordisk A/S of Bagsværd, Denmark, have each been approved by numerous regulatory authorities, including the United States Food and Drug Administration (U.S. FDA) and European Medicines Agency (EMA) for the treatment of patients suffering from type 2 diabetes. These marketed GLP-1 receptor agonists were developed and formulated for injectable and/or oral administration to patients. However, patient adherence to injectable and orally administered therapies for type 2 diabetes is notoriously poor which prohibits many patients from realizing a full and lasting therapeutic potential of GLP-1 receptor agonists. Many patients skip or cease periodic self-administrations of prescribed injectable and orally administered GLP-1 receptor agonists and thus fail to adequately treat and control their own type 2 diabetic condition.

Accordingly, there remains a need for improved GLP-1 receptor agonist polypeptides with high potencies against the GLP-1 receptor, having excellent physicochemical properties, and that are tailored for delivery to patients via more reliable modes of administration than injectable and orally administered therapies.

SUMMARY

It has now been found that compounds of this invention, and pharmaceutically acceptable compositions thereof, are effective as GLP-1 receptor agonists. Such compounds have a general formula as follows:

An isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 200: HX₂X₃GTX₆X₇X₈X₉X₁₀SX₁₂X₁₃X₁₄EX₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FIX₂₄WLKX₂₈GGPX₃₂SGAPP PS-(OH/NH₂) (SEQ ID NO: 200);

wherein each variable is as defined and described herein.

Compounds of the present invention have been designed to attain long elimination half-lives (t_1/2) and are thus described herein as “long-acting” GLP-1 receptor agonists.

Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful for treating a variety of diseases, disorders or conditions, associated with the GLP-1 receptor. Such diseases, disorders, or conditions include metabolic diseases or disorders such as type 2 diabetes, obesity and the need to attain weight loss. In certain embodiments, the invention also relates to methods of treating nonalcoholic fatty liver disease (NAFLD) and/or nonalcoholic steatohepatitis (NASH).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts chronic weight loss (Day 27) in Long Evans (LE) diet-induced obese (DIO) rats treated with exemplary long acting GLP-1 analog polypeptides.

FIG. 2A illustrates comparative in vivo weight loss potencies in DIO rats for compound A120 vs semaglutide (% baseline vs vehicle over 28 days, n=6/dose). Ozempic® (semaglutide, Novo Nordisk A/S of Bagsværd, Denmark) has been approved by the U.S. FDA for the treatment of type 2 diabetes.

FIG. 2B illustrates comparative in vivo type 2 diabetic potencies (i.e., HbA1c lowering) in DIO rats for compound A120 vs semaglutide (A-HbA1c vs vehicle over 28 days, n=9/dose). Antidiabetic potencies were compared against a ZDF rat model for type 2 diabetes.

FIG. 3 depicts a cross-sectional diagram of a representative osmotic mini-pump for drug delivery.

FIG. 4 depicts the potency shift of conjugated GLP-1 receptor agonists by human serum albumin (HSA) binding.

FIG. 5 depicts the change in plasma concentration of compound A120 in rats and monkeys following intravenous and subcutaneous administration.

FIG. 6 depicts dose and concentration response of body weight loss in male Long Evans (LE) Diet Induced Obesity (DIO) rats treated with compound A120 for 27 days. Male LE DIO rats at 18 weeks of age (14 weeks on high fat diet) were dosed (SC; every other day) with six doses of compound A120 ranging from 1 to 300 mcg/kg/day (n=6 animals/dose). Shaded and open circles represent replicate doses (3, 30, 300 mcg/kg/day) of semaglutide from two separate studies.

FIG. 7 depicts semaglutide- and A120-induced weight loss in male LE DIO rats over a 27-day period.

FIG. 8 depicts semaglutide- and A120-induced changes in body composition in male LE DIO rats, highlighting the fact that changes in body composition were primarily due to fat mass loss (body composition was determined on day 26 using quantitative magnetic resonance).

FIG. 9 depicts average daily food intake inhibition and cumulative food intake of mice administered varying doses of compound A120 or semaglultide.

FIG. 10 depicts the reduction in body weight of male Zucker Diabetic Fatty (ZDF) rats dosed (SC injection; every other day) with compound A120 or semaglutide.

FIG. 11 depicts semaglutide- and A120-induced changes in body composition in male ZDF rats, highlighting the fact that changes in body composition were primarily due to fat mass loss (body composition was determined on day 25).

DETAILED DESCRIPTION
General Description of Certain Embodiments of the Invention

Compounds of the present invention, and pharmaceutical compositions thereof, are useful as agonists of the GLP-1 receptor, particularly as agonists of the human GLP-1 receptor. This invention also relates to methods of producing and using such compounds, i.e., GLP-1 receptor agonist polypeptides. Compounds of the present invention are long-acting GLP-1 receptor agonists. These GLP-1 receptor agonist polypeptides are particularly useful in methods of treating metabolic diseases or disorders, such as type 2 diabetes, obesity, and in methods of providing weight loss. In certain embodiments, the invention also relates to methods of treating nonalcoholic fatty liver disease (NAFLD) and/or nonalcoholic steatohepatitis (NASH).

In certain embodiments, the present invention provides an isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 200: HX₂X₃GTX₆X₇X₈X₉X₁₀SX₁₂X₁₃X₁₄EX₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FIX₂₄WLKX₂₈GGPX₃₂SGAPPPS-(OH/NH₂) (SEQ ID NO: 200) or a pharmaceutically acceptable salt thereof, wherein:

X₂is A, 2-aminoisobutyric acid (Aib), or G;

X₃is E or N-methyl Glu;

X₆is F or Y;

X₇is S or T;

X₈is diaminopimelic acid (Dap), E, K, N, N-methyl Ser, Q, S, s, or Y;

X₉is D or E;

X₁₀is I, L, N-methyl Leu, or V;

X₁₂is E, K, Q, or S;

X₁₃is Aib, E, K, Q, S, W, or Y;

X₁₄is E, R, I, K, L, M, Q, or Y;

X₁₆is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn);

X₁₇is E, K, or Q;

X₁₈is A, K, S, or Y;

X₁₉is A, K, or V;

X₂₀is E, K, or R;

X₂₁is Aib, E, H, K, L, Q, or Y;

X₂₄is A, Aib, E, K, Q, S, or Y;

X₂₈is D, E, K, N, Q, S, or Y; and

X₃₂is Dap, H, K, R, or S;

wherein when X₁₆is Dab, Dap, K, k, or Orn, it is covalently bound to a lipophilic substituent, optionally via a spacer;

wherein when X₁₆is E, at least one of X₁₄, X₁₇, X₁₈, X₁₉, X₂₀, or X₂₁is selected to be lysine wherein the lysine residue is covalently bound to a lipophilic substituent, optionally via a spacer; and

wherein the isolated polypeptide optionally further comprises two amino acid residues having side chains that are covalently joined to form a bridging moiety.

In some embodiments, the bridging moiety is a lactam bridging moiety. For example, in some embodiments, the isolated polypeptide optionally further comprises two amino acid residues, wherein one is lysine and the other is glutamic acid, and the amino-containing sidechain of lysine and the carboxy-containing sidechain of glutamic acid are covalently joined, with loss of water, to form a lactam bridging moiety. In some embodiments, the lactam bridging moiety is formed via covalent bonds between side chains of a lysine and a glutamic acid at positions X₁₇and X₂₁, respectively; at positions X₂₁and X₁₇, respectively; at positions X₂₁and X₂₈, respectively; at positions X₂₈and X₂₁, respectively; at positions X₂₀and X₂₄, respectively; at positions X₂₄and X₂₀, respectively; or at positions X₁₂and X₁₆, respectively.

In one embodiment, the isolated polypeptide is not an analogue of exenatide, HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(SEQ ID NO: 300), having K₁₂or K₂₇covalently bound to a lipophilic substituent, optionally via a spacer.

In some embodiments, X₂is Aib.

In some embodiments, if X₂₄is S, then X₁₀is V.

In some embodiments, X₁₄is Y.

In some embodiments, X₁₄is M, and X₁₆is K covalently bound to a lipophilic substituent, optionally via a spacer.

In some embodiments, X₁₆is lysine covalently bound to a lipophilic substituent, optionally via a spacer.

In some embodiments, if X₁₆is E, then X₂₁is K or E. In some embodiments, if X₁₆is E, then X₂₁is K. In some embodiments, if X₁₆is E, then X₂₁is E.

In some embodiments, the peptide further comprises a lactam bridge formed via an amide bond between the side chains of a lysine and a glutamic acid at positions X₂₀and X₂₄, respectively.

Definitions

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a solvent” includes a combination of two or more such solvents, reference to “a peptide” includes one or more peptides, or mixtures of peptides, reference to “a drug” includes one or more drugs, reference to “an osmotic delivery device” includes one or more osmotic delivery devices, and the like. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Unless specifically stated or obvious from context, as used herein, the term “substantially” is understood as within a narrow range of variation or otherwise normal tolerance in the art. Substantially can be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0.001% of the stated value.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other methods and materials similar, or equivalent, to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

The terms “drug,” “therapeutic agent,” and “beneficial agent” are used interchangeably to refer to any therapeutically active substance that is delivered to a subject to produce a desired beneficial effect. In one embodiment of the present invention, the drug is a polypeptide. In another embodiment of the present invention, the drug is a small molecule, for example, hormones such as androgens or estrogens. The devices and methods of the present invention are well suited for the delivery of proteins, small molecules and combinations thereof.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and typically refer to a molecule comprising a chain of two or more amino acids (e.g., most typically L-amino acids, but also including, e.g., D-amino acids, modified amino acids, amino acid analogs, and amino acid mimetics).

In some embodiments, naturally-occurring L-amino acids, are represented by either conventional three-letter, or capitalized one-letter, amino acid designations of Table 1. In other embodiments, naturally-occurring L-amino acids and D-amino acids, are both represented by either conventional three-letter, or capitalized one-letter, amino acid designations of Table 1. In still other embodiments, D-amino acids, are represented by lower-case one-letter amino acid designations corresponding to one-letter designations of Table 2, i.e., a, 1, m, f, w, k, q, e, s, p, v, c, y, h, r, n, d, and t.

TABLE 1

Naturally-occurring amino acids

G
Glycine
Gly
P
Proline
Pro

A
Alanine
Ala
V
Valine
Val

L
Leucine
Leu
I
Isoleucine
Ile

M
Methionine
Met
C
Cysteine
Cys

F
Phenylalanine
Phe
Y
Tyrosine
Tyr

W
Tryptophan
Trp
H
Histidine
His

K
Lysine
Lys
R
Arginine
Arg

Q
Glutamine
Gln
N
Asparagine
Asn

E
Glutamic Acid
Glu
D
Aspartic Acid
Asp

S
Serine
Ser
T
Threonine
Thr

TABLE 2

Lower case designations refer to

D stereoisomers of amino acids

p
Proline
D-Pro

a
D-Alanine
D-Ala
v
D-Valine
D-Val

l
D-Leucine
D-Leu
i
D-Isoleucine
D-Ile

m
D-Methionine
D-Met
c
D-Cysteine
D-Cys

f
D-Phenylalanine
D-Phe
y
D-Tyrosine
D-Tyr

w
D-Tryptophan
D-Trp
h
D-Histidine
D-His

k
D-Lysine
D-Lys
r
D-Arginine
D-Arg

q
D-Glutamine
D-Gln
n
D-Asparagine
D-Asn

e
D-Glutamic Acid
D-Glu
d
D-Aspartic Acid
D-Asp

s
D-Serine
D-Ser
t
D-Threonine
D-Thr

Peptides may be naturally occurring, synthetically produced, or recombinantly expressed. Peptides may also comprise additional groups modifying the amino acid chain, for example, functional groups added via post-translational modification. Examples of post-translation modifications include, but are not limited to, acetylation, alkylation (including, methylation), biotinylation, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, phosphopantetheinylation, phosphorylation, selenation, and C-terminal amidation. The term peptide also includes peptides comprising modifications of the amino terminus and/or the carboxy terminus. Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C₁-C₄alkyl). The term peptide also includes modifications, such as but not limited to those described above, of amino acids falling between the amino and carboxy termini. In one embodiment, a peptide may be modified by addition of a small-molecule drug.

The terminal amino acid at one end of the peptide chain typically has a free amino group (i.e., the amino terminus). The terminal amino acid at the other end of the chain typically has a free carboxyl group (i.e., the carboxy terminus). Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminus and increasing in the direction of the carboxy terminus of the peptide.

The phrase “amino acid residue” as used herein refers to an amino acid that is incorporated into a peptide by an amide bond or an amide bond mimetic.

The term “insulinotropic” as used herein typically refers to the ability of a compound, e.g., a peptide, to stimulate or affect the production and/or activity of insulin (e.g., an insulinotropic hormone). Such compounds typically stimulate or otherwise affect the secretion or biosynthesis of insulin in a subject. Thus, an “insulinotropic peptide” is an amino acid-containing molecule capable of stimulating or otherwise affecting secretion or biosynthesis of insulin.

The term “insulinotropic peptide” as used herein includes, but is not limited to, glucagon-like peptide 1 (GLP-1), as well as derivatives and analogues thereof, GLP-1 receptor agonists, such as exenatide, exenatide having the amino acid sequence of SEQ ID NO: 300, as well as derivatives and analogues thereof.

The term “acylated” as used herein, in relation to disclosed polypeptides, means the disclosed polypeptide is substituted with one or more lipophilic substituents each optionally via a spacer, wherein “lipophilic substituent” and “spacer” are defined herein. Certain lipophilic substituents, each optionally via a spacer, can bind albumin and confer affinity to albumin to the resulting acylated polypeptide. The extent is variable, and depending on numerous factors, to which lipophilic substituents, each optionally via a spacer, bind albumin and confer affinity to albumin to the resulting acylated polypeptide. Numerous factors include identities of the lipophilic substituent, optional spacer, polypeptide, and the site of covalent attachment to the polypeptide.

The terms “linear” or “liner polypeptide” as used herein, refer to a “non-acylated” polypeptide, in other words, a disclosed GLP-1 receptor agonist polypeptide without a lipophilic substituents each optionally via a spacer, wherein “lipophilic substituent” and “spacer” are defined herein.

The terms “conjugated” or conjugated polypeptide” as used herein, refer to an “acylated” polypeptide, in other words, a disclosed GLP-1 receptor agonist polypeptide having one or more lipophilic substituents each optionally via a spacer, wherein “lipophilic substituent” and “spacer” are defined herein.

As used herein, the term “pharmaceutically acceptable salts” refers to derivatives of the disclosed polypeptides wherein the parent polypeptide is modified by converting an existing acid or base moiety to its salt form. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

The term “vehicle” as used herein refers to a medium used to carry a compound, e.g., a drug or a particle containing a drug. Vehicles of the present invention typically comprise components such as polymers and solvents. The suspension vehicles of the present invention typically comprise solvents and polymers that are used to prepare suspension formulations further comprising drug particle formulations.

The phrase “phase separation” as used herein refers to the formation of multiple phases (e.g., liquid and gel phases) in the suspension vehicle, such as when the suspension vehicle contacts the aqueous environment. In some embodiments of the present invention, the suspension vehicle is formulated to exhibit phase separation upon contact with an aqueous environment having less than approximately 10% water.

The phrase “single-phase” as used herein refers to a solid, semisolid, or liquid homogeneous system that is physically and chemically uniform throughout.

The term “dispersed” as used herein refers to dissolving, dispersing, suspending, or otherwise distributing a compound, for example, a drug particle formulation, in a suspension vehicle.

The phrase “chemically stable” as used herein refers to formation in a formulation of an acceptable percentage of degradation products produced over a defined period of time by chemical pathways, such as deamidation (usually by hydrolysis), aggregation, or oxidation.

The phrase “physically stable” as used herein refers to formation in a formulation of an acceptable percentage of aggregates (e.g., dimers and other higher molecular weight products). Further, a physically stable formulation does not change its physical state as, for example, from liquid to solid, or from amorphous to crystal form.

The term “viscosity” as used herein typically refers to a value determined from the ratio of shear stress to shear rate (see, e.g., Considine, D. M. & Considine, G. D., Encyclopedia of Chemistry, 4th Edition, Van Nostrand, Reinhold, N.Y., 1984) essentially as follows:

F/A=μ*V/L (Equation 1)

- where F/A=shear stress (force per unit area),
- μ=a proportionality constant (viscosity), and
- V/L=the velocity per layer thickness (shear rate).

From this relationship, the ratio of shear stress to shear rate defines viscosity. Measurements of shear stress and shear rate are typically determined using parallel plate rheometry performed under selected conditions (for example, a temperature of about 37° C.). Other methods for the determination of viscosity include, measurement of a kinematic viscosity using viscometers, for example, a Cannon-Fenske viscometer, an Ubbelohde viscometer for the Cannon-Fenske opaque solution, or a Ostwald viscometer. Generally, suspension vehicles of the present invention have a viscosity sufficient to prevent a particle formulation suspended therein from settling during storage and use in a method of delivery, for example, in an implantable, drug delivery device.

The term “non-aqueous” as used herein refers to an overall moisture content, for example, of a suspension formulation, typically of less than or equal to about 10 wt %, for example, less than or equal to about 7 wt %, less than or equal to about 5 wt %, and/or less than about 4 wt %.

Also, a particle formulation of the present invention comprises less than about 10 wt %, for example, less than about 5 wt %, residual moisture.

The term “subject” as used herein refers to any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as rhesus macaques and other monkey species and chimpanzees and other ape species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age or gender. Thus, both adult and newborn individuals are intended to be covered.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

The term “osmotic delivery device” as used herein typically refers to a device used for delivery of a drug (e.g., a disclosed GLP-1 receptor agonist polypeptide) to a subject, wherein the device comprises, for example, a reservoir (made, e.g., from a titanium alloy) having a lumen that contains a suspension formulation comprising a drug (e.g., a disclosed GLP-1 receptor agonist polypeptide) and an osmotic agent formulation. A piston assembly positioned in the lumen isolates the suspension formulation from the osmotic agent formulation. A semi-permeable membrane is positioned at a first distal end of the reservoir adjacent the osmotic agent formulation and a diffusion moderator (which defines a delivery orifice through which the suspension formulation exits the device) is positioned at a second distal end of the reservoir adjacent the suspension formulation. Typically, the osmotic delivery device is implanted within the subject, for example, subdermally or subcutaneously (e.g., in the inside, outside, or back of the upper arm and in the abdominal area). An exemplary osmotic delivery device is the DUROS® (ALZA Corporation, Mountain View, Calif.) delivery device. Examples of terms synonymous to “osmotic delivery device” include but are not limited to “osmotic drug delivery device”, “osmotic drug delivery system”, “osmotic device”, “osmotic delivery device”, “osmotic delivery system”, “osmotic pump”, “implantable drug delivery device”, “drug delivery system”, “drug delivery device”, “implantable osmotic pump”, “implantable drug delivery system”, and “implantable delivery system”. Other terms for “osmotic delivery device” are known in the art.

The term “continuous delivery” as used herein typically refers to a substantially continuous release of drug from an osmotic delivery device and into tissues near the implantation site, e.g., subdermal and subcutaneous tissues. For example, an osmotic delivery device releases drug essentially at a predetermined rate based on the principle of osmosis. Extracellular fluid enters the osmotic delivery device through the semi-permeable membrane directly into the osmotic engine that expands to drive the piston at a slow and consistent rate of travel. Movement of the piston forces the drug formulation to be released through the orifice of the diffusion moderator. Thus, release of the drug from the osmotic delivery device is at a slow, controlled, consistent rate.

The term “substantial steady-state delivery” as used herein typically refers to delivery of a drug at or near a target concentration over a defined period of time, wherein the amount of the drug being delivered from an osmotic delivery device is substantially zero-order delivery.

Substantial zero-order delivery of an active agent (e.g., a disclosed GLP-1 receptor agonist polypeptide) means that the rate of drug delivered is constant and is independent of the drug available in the delivery system; for example, for zero-order delivery, if the rate of drug delivered is graphed against time and a line is fitted to the data the line has a slope of approximately zero, as determined by standard methods (e.g., linear regression).

The phrase “drug half-life” as used herein refers how long it takes a drug to be eliminated from blood plasma by one half of its concentration. A drug's half-life is usually measured by monitoring how a drug degrades when it is administered via injection or intravenously. A drug is usually detected using, for example, a radioimmunoassay (MA), a chromatographic method, an electrochemiluminescent (ECL) assay, an enzyme linked immunosorbent assay (ELISA) or an immunoenzymatic sandwich assay (IEMA).

The terms “μg” and “mcg” and “ug” are understood to mean “micrograms”. Similarly, the terms “μl” and “uL” are understood to mean “microliter”, and the terms “μM” and “uM” are understood to mean “micromolar”.

The term “serum” is meant to mean any blood product from which a substance can be detected. Thus, the term serum includes at least whole blood, serum, and plasma. For example, “an amount of [a substance] in a subject's serum” would cover “an amount of [the substance] in a subject's plasma”.

Baseline is defined as the last assessment on or before the day of the initial placement of an osmotic delivery device (containing drug or placebo).

Endogenous GLP-1, GLP-1 Receptors, and Certain GPL-1 Analogs

The phrase “incretin mimetics” as used herein includes, but is not limited to GLP-1 peptide, GLP-1 (7-36), GLP-1 receptor agonists, peptide derivatives of GLP-1, peptide analogs of GLP-1, exenatide, exenatide having the amino acid sequence of exendin-4 (the naturally-occurring form of exenatide, exenatide-LAR, lixisenatide, liraglutide, semaglutide, dulaglutide, albiglutide, and taspoglutide. Incretin mimetics are also referred to herein as “insulinotropic peptides.” Incretin mimetics which target the GLP-1 receptor are also known in the literature as “GLP-1 receptor agonists” or “GLP-1 agonists,” with both terms being used interchangeably herein.

The term “GLP-1” refers to a polypeptide, glucagon-like peptide-1(7-36)amide, a 30-residue peptide hormone released from intestinal L cells following nutrient consumption. GLP-1 has the amino acid sequence of (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂), SEQ ID NO: 301. GLP-1 is a regulatory peptide that binds to the extracellular region of the GLP-1 receptor (GLP-1R), a G-coupled protein receptor on β cell and via adenyl cyclase activity and production of cAMP stimulates the insulin response to the nutrients that are absorbed from the gut [Baggio 2007, “Biology of incretins: GLP-1 and GIP,” Gastroenterology, vol. 132(6):2131-57; Holst 2008, “The incretin system and its role in type 2 diabetes mellitus,” Mol Cell Endocrinology, vol. 297(1-2):127-36]. The effects of GLP-1R agonism are multiple. GLP-1 maintains glucose homeostasis by enhancing endogenous glucose dependent insulin secretion, rendering the β cells glucose competent and sensitive to GLP-1, suppressing glucagon release, restoring first and second phase insulin secretion, slowing gastric emptying, decreasing food intake, and increasing satiety [Holst 2008 Mol. Cell Endocrinology; Kjems 2003 “The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects,” Diabetes, vol. 52(2): 380-86; Holst 2013 “Incretin hormones and the satiation signal,” Int J Obes (Lond), vol. 37(9):1161-69; Seufert 2014, “The extra-pancreatic effects of GLP-1 receptor agonists: a focus on the cardiovascular, gastrointestinal and central nervous systems,” Diabetes Obes Metab, vol. 16(8): 673-88]. The risk of hypoglycemia is minimal given the mode of action of GLP-1. Glucagon-like peptide-1(7-36)amide (GLP-1) is a 30-residue peptide hormone released from intestinal L cells following nutrient consumption. It potentiates the glucose-induced secretion of insulin from pancreatic beta cells, increases insulin expression, inhibits beta-cell apoptosis, promotes beta-cell neogenesis, reduces glucagon secretion, delays gastric emptying, promotes satiety and increases peripheral glucose disposal. These multiple effects have generated a great deal of interest in the discovery of long-lasting agonists of the GLP-1 receptor (GLP-1R) in order to treat type 2 diabetes. The term “exenatide” as used herein includes, but is not limited to exenatide, exenatide having the amino acid sequence of (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂), SEQ ID NO: 300, native exendin-4, exenatide peptides, exenatide peptide analogs, and exenatide peptide derivatives.

Certain disclosed GLP-1 receptor agonist polypeptides, including those of Table 4 below, exhibit one or more of: excellent solubility, stability, bioavailability, biological activity and specificity, and longer half-lives than those for endogenous GLP-1 and known GLP-1 receptor agonists. Certain disclosed GLP-1 receptor agonist polypeptides were developed to accommodate less frequent administration than is required for presently marketed GLP-1 receptor agonists. Certain disclosed GLP-1 receptor agonist polypeptides were developed for administration via weekly or monthly injections. Certain disclosed GLP-1 receptor agonist polypeptides were developed for administration via implantation of a delivery device comprising the GLP-1 receptor agonist polypeptide, where the delivery device comprises a dose of the GLP-1 receptor agonist polypeptide of up to 3 months, 6 months, 9 months, one year, 18 months or two years.

Description of Exemplary Embodiments

In certain embodiments, the present invention provides an isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 210: HX₂X₃GTX₆X₇X₈X₉X₁₀SX₁₂X₁₃X₁₄EX₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FIX₂₄WLKX₂₈GGPX₃₂SGAPPPS-(OH/NH₂) (SEQ ID NO: 210) or a pharmaceutically acceptable salt thereof, wherein:

X₂is A, 2-aminoisobutyric acid (Aib), or G;

X₃is E or N-methyl Glu;

X₆is F or Y;

X₇is S or T;

X₈is diaminopimelic acid (Dap), E, K, N, N-methyl Ser, Q, S, s, or Y;

X₉is D or E;

X₁₀is I, L, N-methyl Leu, or V;

X₁₂is E, K, Q, or S;

X₁₃is Aib, E, K, Q, S, W, or Y;

X₁₄is E, R, I, K, L, M, Q, or Y;

X₁₆is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn);

X₁₇is E, K, or Q;

X₁₈is A, K, S, or Y;

X₁₉is A, K, or V;

X₂₀is E, K, or R;

X₂₁is Aib, E, H, K, L, Q, or Y;

X₂₄is A, Aib, E, K, Q, S, or Y;

X₂₈is D, E, K, N, Q, S, or Y; and

X₃₂is Dap, H, K, R, or S;

with the proviso that the isolated polypeptide is not exenatide.

In some embodiments, X₂is Aib.

In some embodiments, if X₂₄is S, then X₁₀is V.

In some embodiments, X₁₄is Y.

In some embodiments, X₁₄is M, and X₁₆is K.

In some embodiments, X₁₆is K.

In some embodiments, if X₁₆is E, then X₂₁is K or E. In some embodiments, if X₁₆is E, then X₂₁is K. In some embodiments, if X₁₆is E, then X₂₁is E.

In some embodiments, X₂is A. In some embodiments, X₂is Aib. In some embodiments, X₂is G.

As used herein, Aib refers alternatively to 2-aminoisobutyric acid, α-aminoisobutyric acid, α-methylalanine or 2-methylalanine.

In some embodiments, X₃is E. In some embodiments, X₃is N-methyl Glu.

In some embodiments, X₆is F. In some embodiments, X₆is Y.

In some embodiments, X₇is S. In some embodiments, X₇is T.

In some embodiments, X₈is Dap. In some embodiments, X₈is E. In some embodiments, X₈is K. In some embodiments, X₈is N. In some embodiments, X₈is N-methyl Ser. In some embodiments, X₈is Q. In some embodiments, X₈is S. In some embodiments, X₈is s. In some embodiments, X₈is Y.

As used herein, Dap refers to diaminopimelic acid.

As used herein, s refers to D-serine.

In some embodiments, X₉is D. In some embodiments, X₉is E.

In some embodiments, X₁₀is I. In some embodiments, X₁₀is L. In some embodiments, X₁₀is N-methyl Leu. In some embodiments, X₁₀is V.

In some embodiments, X₁₂is E. In some embodiments, X₁₂is K. In some embodiments, X₁₂is Q. In some embodiments, X₁₂is S.

In some embodiments, X₁₃is Aib. In some embodiments, X₁₃is E. In some embodiments, X₁₃is K. In some embodiments, X₁₃is Q. In some embodiments, X₁₃is S. In some embodiments, X₁₃is W. In some embodiments, X₁₃is Y.

In some embodiments, X₁₄is E. In some embodiments, X₁₄is R. In some embodiments, X₁₄is I. In some embodiments, X₁₄is K. In some embodiments, X₁₄is L. In some embodiments, X₁₄is M. In some embodiments, X₁₄is Q. In some embodiments, X₁₄is Y.

In some embodiments, X₁₆is Dab. In some embodiments, X₁₆is Dap. In some embodiments, X₁₆is E. In some embodiments, X₁₆is K. In some embodiments, X₁₆is k. In some embodiments, X₁₆is Orn.

As used herein, Dab refers alternately to 2,4-diaminobutanoic acid, 2,4-diaminobutyric acid, or α,γ-diaminobutyric acid.

As used herein, k refers to D-lysine.

As used herein, Orn refers to ornithine.

In some embodiments, X₁₇is E. In some embodiments, X₁₇is K. In some embodiments, X₁₇is Q.

In some embodiments, X₁₈is A. In some embodiments, X₁₈is K. In some embodiments, X₁₈is S. In some embodiments, X₁₈is Y.

In some embodiments, X₁₉is A. In some embodiments, X₁₉is K. In some embodiments, X₁₉is V.

In some embodiments, X₂₀is E. In some embodiments, X₂₀is K. In some embodiments, X₂₀is R.

In some embodiments, X₂₁is Aib. In some embodiments, X₂₁is E. In some embodiments, X₂₁is H. In some embodiments, X₂₁is K. In some embodiments, X₂₁is L. In some embodiments, X₂₁is Q. In some embodiments, X₂₁is Y.

In some embodiments, X₂₄is A. In some embodiments, X₂₄is Aib. In some embodiments, X₂₄is E. In some embodiments, X₂₄is K. In some embodiments, X₂₄is Q. In some embodiments, X₂₄is S. In some embodiments, X₂₄is Y.

In some embodiments, X₂₈is D. In some embodiments, X₂₈is E. In some embodiments, X₂₈is K. In some embodiments, X₂₈is N. In some embodiments, X₂₈is Q. In some embodiments, X₂₈is S. In some embodiments, X₂₈is Y.

In some embodiments, X₃₂is Dap. In some embodiments, X₃₂is H. In some embodiments, X₃₂is K. In some embodiments, X₃₂is R. In some embodiments, X₃₂is S.

In some embodiments, carboxy terminal amino acid, i.e. S₃₉, is —S₃₉—(NH₂). In some embodiments, carboxy terminal amino acid S₃₉is —S₃₉—(OH).

In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include the following:

In some embodiments, X₂is Aib and X₁₀is V. In some embodiments, X₂is Aib and X₁₄is Y. In some embodiments, X₂is Aib and X₁₆is K. In some embodiments, X₂is Aib and X₁₉is V. In some embodiments, X₂is Aib and X₁₉is A. In some embodiments, X₂is Aib and X₂₀is R. In some embodiments, X₂is Aib and X₂₀is K. In some embodiments, X₂is Aib and X₂₁is K. In some embodiments, X₂is Aib and X₂₁is Q. In some embodiments, X₂is Aib and X₂₄is S. In some embodiments, X₂is Aib and X₂₄is E.

In some embodiments, X₁₀is V and X₁₄is Y. In some embodiments, X₁₀is V and X₁₆is K. In some embodiments, X₁₀is V and X₁₉is V. In some embodiments, X₁₀is V and X₁₉is A. In some embodiments, X₁₀is V and X₂₀is R. In some embodiments, X₁₀is V and X₂₀is K. In some embodiments, X₁₀is V and X₂₁is K. In some embodiments, X₁₀is V and X₂₁is Q. In some embodiments, X₁₀is V and X₂₄is S. In some embodiments, X₁₀is V and X₂₄is E.

In some embodiments, X₁₄is Y and X₁₆is K. In some embodiments, X₁₄is Y and X₁₉is V. In some embodiments, X₁₄is Y and X₁₉is A. In some embodiments, X₁₄is Y and X₂₀is R. In some embodiments, X₁₄is Y and X₂₀is K. In some embodiments, X₁₄is Y and X₂₁is K. In some embodiments, X₁₄is Y and X₂₁is Q. In some embodiments, X₁₄is Y and X₂₄is S. In some embodiments, X₁₄is Y and X₂₄is E.

In some embodiments, X₁₆is K and X₁₉is V. In some embodiments, X₁₆is K and X₁₉is A. In some embodiments, X₁₆is K and X₂₀is R. In some embodiments, X₁₆is K and X₂₀is K. In some embodiments, X₁₆is K and X₂₁is K. In some embodiments, X₁₆is K and X₂₁is Q. In some embodiments, X₁₆is K and X₂₄is S. In some embodiments, X₁₆is K and X₂₄is E.

In some embodiments, X₁₉is V and X₂₀is R. In some embodiments, X₁₉is V and X₂₀is K. In some embodiments, X₁₉is V and X₂₁is K. In some embodiments, X₁₉is V and X₂₁is Q. In some embodiments, X₁₉is V and X₂₄is S. In some embodiments, X₁₉is V and X₂₄is E.

In some embodiments, X₁₉is A and X₂₀is R. In some embodiments, X₁₉is A and X₂₀is K. In some embodiments, X₁₉is A and X₂₁is K. In some embodiments, X₁₉is A and X₂₁is Q. In some embodiments, X₁₉is A and X₂₄is S. In some embodiments, X₁₉is A and X₂₄is E.

In some embodiments, X₂₀is R and X₂₁is K. In some embodiments, X₂₀is R and X₂₁is Q. In some embodiments, X₂₀is R and X₂₄is S. In some embodiments, X₂₀is R and X₂₄is E.

In some embodiments, X₂₀is K and X₂₁is K. In some embodiments, X₂₀is K and X₂₁is Q. In some embodiments, X₂₀is K and X₂₄is S. In some embodiments, X₂₀is K and X₂₄is E.

In some embodiments, X₂₁is K and X₂₄is S. In some embodiments, X₂₁is K and X₂₄is E. In some embodiments, X₂₁is Q and X₂₄is S. In some embodiments, X₂₁is Q and X₂₄is E.

In some embodiments, certain amino acids represented by the consensus sequence of SEQ ID NO: 210 include the following:

In some embodiments, X₂is Aib, X₁₀is V, and X₁₄is Y. In some embodiments, X₂is Aib, X₁₀is V, and X₁₆is K. In some embodiments, X₂is Aib, X₁₀is V, and X₁₉is V. In some embodiments, X₂is Aib, X₁₀is V, and X₁₉is A. In some embodiments, X₂is Aib, X₁₀is V, and X₂₀is R. In some embodiments, X₂is Aib, X₁₀is V, and X₂₀is K. In some embodiments, X₂is Aib, X₁₀is V, and X₂₁is K. In some embodiments, X₂is Aib, X₁₀is V, and X₂₁is Q. In some embodiments, X₂is Aib, X₁₀is V, and X₂₄is S. In some embodiments, X₂is Aib, X₁₀is V, and X₂₄is E.

In some embodiments, X₂is Aib, X₁₄is Y, and X₁₆is K. In some embodiments, X₂is Aib, X₁₄is Y, and X₁₉is V. In some embodiments, X₂is Aib, X₁₄is Y, and X₁₉is A. In some embodiments, X₂is Aib, X₁₄is Y, and X₂₀is R. In some embodiments, X₂is Aib, X₁₄is Y, and X₂₀is K. In some embodiments, X₂is Aib, X₁₄is Y, and X₂₁is K. In some embodiments, X₂is Aib, X₁₄is Y, and X₂₁is Q. In some embodiments, X₂is Aib, X₁₄is Y, and X₂₄is S. In some embodiments, X₂is Aib, X₁₄is Y, and X₂₄is E.

In some embodiments, X₁₀is V, X₁₄is Y, and X₁₆is K. In some embodiments, X₁₀is V, X₁₄is Y, and X₁₉is V. In some embodiments, X₁₀is V, X₁₄is Y, and X₁₉is A. In some embodiments, X₁₀is V, X₁₄is Y, and X₂₀is R. In some embodiments, X₁₀is V, X₁₄is Y, and X₂₀is K. In some embodiments, X₁₀is V, X₁₄is Y, and X₂₁is K. In some embodiments, X₁₀is V, X₁₄is Y, and X₂₁is Q. In some embodiments, X₁₀is V, X₁₄is Y, and X₂₄is S. In some embodiments, X₁₀is V, X₁₄is Y, and X₂₄is E.

In some embodiments, X₁₄is Y, X₁₆is K, and X₁₉is V. In some embodiments, X₁₄is Y, X₁₆is K, and X₁₉is A. In some embodiments, X₁₄is Y, X₁₆is K, and X₂₀is R. In some embodiments, X₁₄is Y, X₁₆is K, and X₂₀is K. In some embodiments, X₁₄is Y, X₁₆is K, and X₂₁is K. In some embodiments, X₁₄is Y, X₁₆is K, and X₂₁is Q. In some embodiments, X₁₄is Y, X₁₆is K, and X₂₄is S. In some embodiments, X₁₄is Y, X₁₆is K, and X₂₄is E.

In some embodiments, X₁₄is Y, X₁₉is V, and X₂₁is K. In some embodiments, X₁₄is Y, X₁₉is A, and X₂₁is K. In some embodiments, X₁₄is Y, X₂₀is R, and X₂₁is K. In some embodiments, X₁₄is Y, X₂₀is K, and X₂₁is K. In some embodiments, X₁₄is Y, X₂₁is K, and X₂₄is S. In some embodiments, X₁₄is Y, X₂₁is K, and X₂₄is E.

In some embodiments, X₁₆is K, X₁₉is A, and X₂₀is R. In some embodiments, X₁₆is K, X₁₉is A, and X₂₀is K. In some embodiments, X₁₆is K, X₁₉is A, and X₂₁is K. In some embodiments, X₁₆is K, X₁₉is A, and X₂₁is Q. In some embodiments, X₁₆is K, X₁₉is A, and X₂₄is S. In some embodiments, X₁₆is K, X₁₉is A, and X₂₄is E.

In some embodiments, X₁₉is V, X₂₀is R, and X₂₁is K. In some embodiments, X₁₉is V, X₂₀is R, and X₂₁is Q. In some embodiments, X₁₉is V, X₂₀is R, and X₂₄is S. In some embodiments, X₁₉is V, X₂₀is R, and X₂₄is E.

In some embodiments, X₁₉is A, X₂₀is R, and X₂₁is K. In some embodiments, X₁₉is A, X₂₀is R, and X₂₁is Q. In some embodiments, X₁₉is A, X₂₀is R, and X₂₄is S. In some embodiments, X₁₉is A, X₂₀is R, and X₂₄is E.

In some embodiments, X₂₀is R, X₂₁is K, and X₂₄is S. In some embodiments, X₂₀is R, X₂₁is K, and X₂₄is E.

In some embodiments, X₂₀is K, X₂₁is K, and X₂₄is S. In some embodiments, X₂₀is K, X₂₁is K, and X₂₄is E.

X₂is A, 2-aminoisobutyric acid (Aib), or G;

X₃is E or N-methyl Glu;

X₆is F or Y;

X₇is S or T;

X₈is diaminopimelic acid (Dap), E, K, N, N-methyl Ser, Q, S, s, or Y;

X₉is D or E;

X₁₀is I, L, N-methyl Leu, or V;

X₁₂is E, K, Q, or S;

X₁₃is Aib, E, K, Q, S, W, or Y;

X₁₄is E, R, I, K, L, M, Q, or Y;

X₁₆is 2,4-diaminobutanoic acid (Dab), Dap, E, K, k, or ornithine (Orn);

X₁₇is E, K, or Q;

X₁₈is A, K, S, or Y;

X₁₉is A, K, or V;

X₂₀is E, K, or R;

X₂₁is Aib, E, H, K, L, Q, or Y;

X₂₄is A, Aib, E, K, Q, S, or Y;

X₂₈is D, E, K, N, Q, S, or Y; and

X₃₂is Dap, H, K, R, or S;

wherein when X₁₆is Dab, Dap, K, k, or Orn, it is covalently bound to a lipophilic substituent, optionally via a spacer;

wherein the isolated polypeptide optionally further comprises two amino acid residues having side chains that are covalently joined to form a bridging moiety.

In some embodiments, X₂is Aib.

In some embodiments, if X₂₄is S, then X₁₀is V.

In some embodiments, X₁₄is Y.

In some embodiments, X₁₄is M, and X₁₆is K covalently bound to a lipophilic substituent, optionally via a spacer.

In some embodiments, X₁₆is K covalently bound to a lipophilic substituent, optionally via a spacer.