KR102093463B1 - Double-acylated glp-1 derivatives - Google Patents

Abstract

The present invention relates to derivatives of GLP-1 analogs, or pharmaceutically acceptable salts, amides, or esters thereof, the analogs of which are positions 27 of GLP-1 (7-37) (SEQ ID NO: 1). A first K residue at the position corresponding to; Includes a second K residue at a position corresponding to the T position of GLP-1 (7-37), where T is an integer in the range 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is referred to as K ²⁷ and the second K residue is referred to as K ^T ; Its derivatives are K ²⁷ and Includes two albumin binding moieties attached to K ^T via a linker, wherein the albumin binding moieties are from HOOC (CH ₂ ) _x -CO- and HOOC-C ₆ H ₄ -O- (CH ₂ ) _y -CO- A selected extension portion; Where x is an integer in the range 6-16 and y is an integer in the range 3-17; Wherein the linker comprises elements of the formula NH- (CH ₂ ) _2- (O- (CH ₂ ) ₂ ) _k -O- (CH ₂ ) _n -CO-, where k is an integer ranging from 1-5, n is an integer ranging from 1-5. The present invention relates to the corresponding novel GLP-1 analogs as well as their pharmaceutical use, for example in the treatment and / or prevention of all forms of diabetes and related diseases. Derivatives are suitable for oral administration.

Description

Double acylated GLP-1 derivative {DOUBLE-ACYLATED GLP-1 DERIVATIVES}

Cross-reference of related applications

This application is a 37 C.F.R. US Provisional Patent Application No. 61/474,913, filed April 13, 2011 under §1.53(c), is hereby incorporated by reference in its entirety.

Technical field

The present invention relates to a derivative of an analogue of glucagon-like peptide 1 (GLP-1), more specifically, K ²⁷ of the peptide and It relates to double acylated GLP-1 derivatives acylated at other K residues, and their pharmaceutical use.

Inclusion by reference in the sequence listing

The sequence listing titled “SEQUENCE LISTING” is 568 bytes, was created on April 11, 2012, and is incorporated herein by reference.

Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. 1664-669 discloses derivatives of GLP-1 (7-37) comprising some double acylated.

WO 98/08871 A1 discloses a number of GLP-1 derivatives, including some double acylated. Liraglutide, a single acylated GLP-1 derivative sold in 2009 by Novo Nordisk A/S and for once daily administration is also disclosed in WO 98/08871 A1 (Example 37).

WO 99/43706 A1 has any K ^27,26 and Numerous single and double acylated GLP-1 derivatives are ^disclosed , including K 27,34 derivatives.

WO 06/097537 A2 discloses a number of GLP-1 derivatives, including semaglutide (Example 4), a single acylated GLP-1 derivative for once weekly administration under development by Novo Nordisk A/S. .

Angewandte Chemie International Edition 2008, vol. 47, p. 3196-3201 reports the discovery and characterization of a class of 4-(p-iodophenyl)butyric acid derivatives that intentionally exhibit stable non-covalent interactions with both mouse serum albumin (MSA) and human serum albumin (HSA).

The present invention relates to derivatives of the GLP-1 peptide.

The derivative is acylated at the lysine substituted for the native glutamic acid at position 27, as well as at other lysine residues. The side chain is the albumin binding moiety. They preferably comprise an extended moiety selected from fatty diacids and fatty acids having all optionally substituted terminal phenoxy groups. The carboxy group of the fatty acid or fatty diacid is preferably acylated via a linker, preferably from its epsilon-amino group to the lysine residue of the GLP-1 peptide.

GLP-1 peptide compared to GLP-1 (7 to 37), for example, one or more additions, one or more deletions and / or one or more substitutions, GLP with a total of 10 amino acid differences It may be an analog of -1 (7 to 37) (SEQ ID NO: 1).

More specifically, in the present invention, the analogue is the first K residue at the position corresponding to the 27 position of GLP-1 (7-37) (SEQ ID NO: 1); A derivative of a GLP-1 analog comprising a second K moiety at a position corresponding to the T position of GLP-1 (7-37), or a pharmaceutically acceptable salt, amide, or ester thereof, wherein T Is an integer in the range 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Where the first K residue is K²⁷And the second K residue is K^TIs referred to as; Each of its derivatives is K²⁷And K^TIt comprises two albumin binding moieties attached to through a linker, wherein each albumin binding moiety is HOOC(CH₂)_x-CO-* and HOOC-C₆H₄-O-(CH₂)_y-CO-*, wherein x is an integer in the range of 6-16 and y is an integer in the range of 3-17, wherein the linker is the formula Chem. It contains 5 linker elements:

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

The present invention is particularly for use in the treatment and/or prevention of all forms of diabetes and related diseases such as dietary disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression, and for use as such agents.

Furthermore, the present invention relates to intermediate products in the form of novel GLP-1 analogues associated with the preparation of certain derivatives of the present invention.

The derivatives of the present invention are biologically active. Also, or alternatively, they have an extended pharmacokinetic profile. Also, or alternatively, they are stable against digestion by enzymes in the gastrointestinal tract. Also, or alternatively, they have high oral bioavailability. These properties are important in the development of the next generation of GLP-1 compounds for subcutaneous, intravenous, and/or particularly oral administration.

In the following, the Greek letters are their symbols or corresponding written names, for example: α = alpha; β = beta; ε = epsilon; γ = gamma; ω = omega; It can be represented by the like. In addition, the Greek letter of μ can be represented by “u”, for example μl=ul, or μM=uM.

An asterisk (*) in the formula refers to i) the point of attachment, ii) a radical, and/or iii) an unshared electron.

In a first aspect, the present invention relates to a derivative of a GLP-1 analog, or a pharmaceutically acceptable salt, amide, or ester thereof, the analog of which is GLP-1(7-37) (SEQ ID NO: A first K residue at a position corresponding to position 27 of 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;} Its derivatives are K ²⁷ and respectively It ^{comprises two albumin binding moieties attached to K T} through a linker, wherein the albumin binding moiety is described in Chem. 1 and Chem. It includes an extended portion selected from two:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where x is an integer in the range of 6-16, y is an integer in the range of 3-17, and the linker is selected from Chem. Contains 5:

Chem. 5:

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

GLP-1 analog

As used herein, the term “GLP-1 analog” or “analog of GLP-1” refers to a peptide or compound that is a variant of human glucagon-like peptide-1 (GLP-1(7-37)), as SEQ ID NO: 1 Refers to a sequence included in the sequence listing. Peptides having the sequence of SEQ ID NO: 1 may also be referred to as “native” GLP-1.

In the sequence listing, the first amino acid residue (histidine) of SEQ ID NO: 1 is designated as number 1. However, in the following according to art-established practice, this histidine residue is referred to as number 7 and thus the subsequent amino acid residue is numbered, ending with glycine number 37. Thus, generally, by reference herein, the amino acid residue number or position number of the GLP-1 (7-37) sequence begins with His at position 7 and ends with Gly at position 37.

GLP-1 analogues of the derivatives of the invention include i) the number of amino acid residues in the original GLP-1 (7-37) (i.e., the corresponding positions in the original GLP-1) corresponding to the changed amino acid residues, and ii) the actual It can be explained with reference to change. The following are non-limiting examples of suitable analog nomenclature.

A non-limiting example of a GLP-1 analog of the derivative of the present invention is an analog modified to include a first lysine residue at the position corresponding to position 27 of GLP-1 (7-37), and a second lysine residue at position 12. Otherwise the amino acid sequence of this analog is identical to the original GLP-1, and this analog ^{can be referred to as K 12} ,K ²⁷ -GLP-1 (7-37). This designation refers to the amino acid sequence of the original GLP-1 in which phenylalanine at position 12 is substituted with lysine and glutamic acid at position 27 is substituted with lysine.

The GLP-1 analog-forming portion of the derivatives of the present invention contains up to 10 amino acid changes compared to the original GLP-1 (7-37) (SEQ ID NO: 1). That is, it is a GLP-1 (7-37) peptide in which a number of amino acid residues have been altered when compared to the original GLP-1 (7-37) (SEQ ID NO: 1). These changes may independently represent one or more amino acid substitutions, additions, and/or deletions.

The following are non-limiting examples of suitable analog nomenclature.

For example, the analog [Aib8,Lys22,Val25,Arg26,Lys27,His31,Arg34]-GLP-1-(7-37) is the Aib(α-amino Isobutyric acid), glycine to lysine at position 22, alanine to valine at position 25, lysine to arginine at position 26, glutamic acid to lysine at position 27, tryptophan at position 31 GLP-1 (7-37) peptide having a substitution of histidine of and a substitution of lysine to arginine at position 34. This analog can also be simply referred to as (8Aib, 22K, 25V, 26R, 27K, 31H, 34R).

As another example, the analog [Aib8,Lys20,Glu22,Arg26,Lys27,Glu30,Gly34]-GLP-1-(7-34) is the substitution of alanine to Aib at position 8 as compared to the original GLP-1, 20 Leucine to lysine at position, glycine to glutamic acid at position 22, lysine to arginine at position 26, glutamic acid to lysine at position 27, alanine to glutamic acid at position 30, position 34 GLP-1(7-37) peptide changed by the substitution of lysine in glycine and by deletion of the C-terminus of glycine-arginine-glycine at positions 35-36-37. This analog can also be referred to simply as (8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37), which is meant by reference to GLP-1 (7-37) and "des" represents a deletion. have.

As a further example, Glu ³⁸ And Analogs comprising Gly ³⁹ include the addition of a dipeptide of (glutamic acid-glycine) to the C-terminus of GLP-1 (7-37) when compared to the original GLP-1, GLP-1 (7- 37) Refers to peptides. This analog can also be briefly mentioned as including (38E, 39G), which is meant by reference to GLP-1 (7-37).

Analogs that “comprise” certain specified changes may contain additional changes when compared to SEQ ID NO: 1. One non-limiting example of an analog comprising (38E, 39G) is Chem. It is the peptide portion of 51.

As is evident from the above example, amino acid residues can be identified by their full name, their single letter code, and/or three letter code. All three of these methods are the same.

The expression “same position” or “corresponding position” characterizes the site of change in the variant GLP-1 (7-37) sequence by reference to the original GLP-1 (7-37) (SEQ ID NO: 1). Can be used to do. The same or corresponding position as well as the number of changes are easily inferred, for example by simple handwriting and eyeballing; And/or standard protein or peptide alignment programs such as the Needleman-Wunsch alignment “alignment” can be used. The algorithm is described in Needleman, S.B. and Wunsch, CD, (1970), Journal of Molecular Biology, 48: 443-453, and the alignment program in Myers and W. Miller's "Optimal Alignments in Linear Space" CABIOS (computer use in life sciences) (1988). It is described in 4:11-17. For alignment, the default scoring matrix BLOSUM50 and the default identity matrix can be used, the penalty for the first residue in the gap is -12, or preferably -10, the penalty for additional residues in the gap is -2, or preferably It can be set to -0.5.

An example of such an alignment is inserted below, where SEQ ID NO: 1 (SEQ_ID_NO_1) is SEQ ID NO: 1, and SEQ ID NO: 2 (analog) is its analogue (22K, 26R, 27K, 30E, 34G, des35-37). :

In cases where non-native amino acids such as Aib are included in the sequence, they can be replaced with X for alignment purposes. If desired, X can be manually corrected later.

As used in the text of the GLP-1 analogues of the derivatives of the invention, for example, the term "peptide" refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds.

The peptides of the present invention comprise at least five constituent amino acids linked by peptide bonds. In certain embodiments the peptide comprises at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 25, or most preferably at least 28 amino acids.

In certain embodiments, the peptide consists of at least 5 constituent amino acids, preferably consists of at least 10, at least 15, at least 20, at least 25, or most preferably consists of at least 28 amino acids.

In a further specific embodiment, the peptide a) consists of, or b) consists of i) 28, ii) 29, iii) 30, iv) 31, v) 32, or vi) 33 amino acids. .

In a further specific embodiment the peptide consists of amino acids interconnected by peptide bonds.

Amino acids are molecules containing amine groups and carboxylic acid groups and optionally one or more additional groups often referred to as side chains.

The term “amino acid” refers to proteinaceous amino acids (encoded by the genetic code comprising the original amino acids and standard amino acids), as well as non- It also includes proteinaceous and synthetic amino acids. Thus, the amino acids can be selected from the group of proteomic amino acids, non-proteinogenic amino acids, and/or synthetic amino acids.

Non-limiting examples of amino acids that are not encoded by the genetic code are gamma-carboxyglutamate, ornithine, and phosphoserine. Non-limiting examples of synthetic amino acids include D-isomers of amino acids such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), β-alanine, and des-amino-histidine (desH, alternative name imidazopropionic acid). , Abbreviated as Imp).

In the following, all amino acids to which no optical isomer is mentioned are understood to mean the L-isomer (unless otherwise specified).

The GLP-1 derivatives and analogs of the present invention have GLP-1 activity. This term refers to the ability to bind to the GLP-1 receptor and initiate signaling pathways, resulting in insulin secretion or other physiological effects known in the art. For example, analogs and derivatives of the present invention can be tested for GLP-1 activity using the assay described in Example 33 herein. The GLP-1 receptor binding assay described in Example 34 herein can also be used to measure GLP-1 activity (low HSA experiment).

GLP-1 derivative

The term “derivative” as used herein in the context of a GLP-1 peptide or analog refers to a chemically modified GLP-1 peptide or analog to which one or more substituents are covalently attached to the peptide. Substituents are also referred to as side chains.

In certain embodiments, due to the fact that the aggregates of the GLP-1-derivative and albumin only slowly disintegrate to release the active pharmaceutical ingredient, the side chains form non-covalent aggregates with albumin, thereby circulating the derivative into the bloodstream. And may also have the effect of prolonging the time of action of the derivative. Thus, the substituents or side chains as a whole are preferably referred to as albumin binding moieties.

In other specific embodiments the albumin binding moiety includes a moiety specifically related to albumin binding and thus extension, which moiety may be referred to as an extension moiety. The extension moiety may be at or near the opposite end of the albumin binding moiety, relative to the point of attachment to the peptide.

In a further specific embodiment the albumin binding moiety comprises a moiety between the extension moiety and the point of attachment to the peptide, which moiety may be referred to as a linker, linker moiety, spacer, and the like. The linker may be optional, and as such, the albumin binding moiety may be the same as the extension moiety in such case.

In certain embodiments, the albumin binding moiety and/or extension moiety is lipophilic and/or negatively charged at physiological pH (7.4).

The albumin binding moiety, extension moiety, or linker may be covalently attached to the lysine residue of the GLP-1 peptide by acylation.

In a preferred embodiment, the active ester of the albumin binding moiety, preferably comprising an extension moiety and a linker, is the amino group of the lysine moiety, preferably its epsilon amino group under the formation of an amide bond (this process is referred to as acylation). Linked sharedly to.

Unless otherwise stated, when a reference is made by acylation of a lysine residue, it is understood to be its epsilon-amino group.

At the first and second K residues (e.g., K ²⁷ and Derivatives comprising two extension moieties attached to K ^T ) via a linker are, for example, acylated twice at the epsilon-amino group of the first and second lysine residues at positions 27 and T of the GLP-1 peptide, respectively, double It may be referred to as an acylation, or dual acylated derivative.

For the purposes of the present invention, the terms "albumin binding moiety", "extended moiety", and "linker" may include reacted as well as unreacted forms of these molecules. Whether one or the other form is meaningful becomes clear from the context in which the term is used.

In one embodiment, each extension moiety is Chem. 1 and Chem. Comprising or consisting of an extension portion independently selected from two:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where x is an integer in the range 6-16 and y is an integer in the range 3-17.

In one embodiment, *(CH ₂ ) _x -* refers to straight or branched, preferably straight, alkylene, where x is an integer in the range 6-16.

In another embodiment, *(CH ₂ ) _y -* refers to straight or branched, preferably straight, alkylene, where y is an integer in the range 3-17.

The term "fatty acid" refers to an aliphatic monocarboxylic acid having 4 to 28 carbon atoms, which is preferably unbranched, and/or even numbered, which may be saturated or unsaturated.

The term "fatty diacid" refers to a fatty acid as defined above but having an additional carboxylic acid group at the omega position. Thus, the fatty diacid is a dicarboxylic acid.

The nomenclature is common in the art, for example, in the above formulas, not only HOOC-* but also *COOH are carboxy; *-C ₆ H ₄ -* is phenylene; *OC-* as well as *CO* are carbonyl (O=C< _** ); C ₆ H ₅ -O* refers to phenoxy. In certain embodiments, aromatic and phenylline radicals such as phenoxy can be independently ortho, meta or para.

As explained above, the GLP-1 derivatives of the present invention are double acylated, ie two albumin binding moieties are covalently attached to the GLP-1 peptide.

In certain embodiments, the two albumin binding moieties (ie the entire side chain) are similar, preferably substantially identical, or most preferably identical.

In other specific embodiments, the two extending portions are similar, preferably substantially identical, or most preferably identical.

In a further specific embodiment, the two linkers are similar, preferably substantially identical, or most preferably identical.

The term “substantially identical” refers to the formation of one or more salts, esters, and/or amides; Preferably the formation of one or more salts, methyl esters, and simple amides; More preferably the formation of no more than two salts, methyl esters, and/or simple amides; Even more preferably the formation of no more than one salt, methyl ester, and/or simple amide; Or, most preferably, differences in identity due to the formation of one or less salts.

In the context of compounds such as albumin binding moieties, extension moieties, and linkers, similarity and/or identity can be determined using any suitable computer program and/or algorithm known in the art.

For example, the similarity of two extension moieties, two linkers, and/or two whole side chains can be suitably determined using molecular fingerprinting. Fingerprints are a mathematical method of representing chemical structures (see, for example, Chemoinformatics: A textbook, Johann Gasteiger and Thomas Engel (Eds), Wiley-VCH Verlag, 2003).

Examples of suitable fingerprints include, without limitation, an ECFP fingerprint such as a unit Y fingerprint, an MDL fingerprint and/or an ECFP_6 fingerprint (ECFP means an extended connection fingerprint).

In certain embodiments, the two extension moieties, two linkers, and/or two entire side chains comprise a) an ECFP_6 fingerprint; b) Unit Y fingerprint; And/or c) an MDL fingerprint.

The Tanimoto coefficient is preferably used to calculate the similarity of two fingerprints for which a), b) or c) is used.

In certain embodiments, when a), b), or c) is used, each of the two extension moieties, two linkers, and/or two total side chains is at least 0.5 (50%); Preferably at least 0.6 (60%); More preferably at least 0.7 (70%), or at least 0.8 (80%); Even more preferably at least 0.9 (90%); Or most preferably at least 0.99 (99%) similarity, such as 1.0 (100%) similarity.

Unit Y fingerprints can be calculated using the program SYBYL (available from Tripos, 1699 South Hanley Road, St. Louis, MO 63144-2319 USA). ECFP_6 and MDL fingerprints can be calculated using the program Pipeline Pilot (available from Accelrys Inc., 10188 Telesis Court, Suite 100, San Diego, CA 92121, USA).

For more details, see for example J. Chem. Inf. Model. 2008, 48, 542-549; J. Chem. Inf. Comput. Sci. 2004, 44, 170-178; J. Med. Chem. 2004, 47, 2743-2749; J. Chem. Inf. Model. 2010, 50, 742-754; As well as SciTegic Pipeline Pilot Chemistry Collection: Basic Chemistry User Guide, March 2008, SciTegic Pipeline Pilot Data Modeling Collection, 2008-both from Accelrys Software Inc., San Diego, US, and guide http://www.tripos. See com/tripos_resources/fileroot/pdfs/Unity_111408.pdf, and http://www.tripos.com/data/SYBYL/SYBYL_072505.pdf.

An example of the similarity calculation is inserted below, Chem. The entire side chain of 66 was compared to its methyl ester, ie the single methyl ester of the glutamine linker moiety (Chem. 66a):

Chem. 66a:

a) the similarity using the ECFP_6 fingerprint is 0.798, b) the similarity using the unit Y fingerprint is 0.957; The similarity using the MDL fingerprint is 0.905.

In the case of two identical side chains (albumin binding moieties), the derivative may be referred to as symmetric.

In certain embodiments, the similarity coefficient is at least 0.80, preferably at least 0.85, more preferably at least 0.90, even more preferably at least 0.95, or most preferably at least 0.99.

Each of the two linkers of the derivatives of the invention may comprise the following first linker elements:

Chem. 5:

,

,

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

In certain embodiments, when k=1 and n=1, this linker element may be referred to as the designated OEG, or the 8-amino-3,6-dioxaoctanoic acid of a di-radical, and/or it is It can be represented by the formula:

Chem. 5a:

*NH-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-CH ₂ -CO-*.

In another specific embodiment, each linker of a derivative of the invention is independently a second linker element, preferably Chem. 6 and/or Chem. Glu di-radicals such as 7:

Chem. 6:

Chem. 7:

,

,

Here, the Glu di-radical may be included p times, where p is an integer in the range of 1-2.

Chem. 6 may also be referred to as gamma-Glu, or simply gGlu, due to the fact that it is the gamma carboxy group of the amino acid glutamic acid that can be used herein to other linker elements or for linking of lysine to the epsilon-amino group. As explained above, other linker elements can be, for example, other Glu moieties, or OEG molecules. The amino group of Glu may in turn form an amide bond with the carboxy group of the extended moiety, or, for example, the carboxy group of the OEG molecule, if present, or, for example, the gamma-carboxy group of other Glu, if present.

Chem. 7 may also be referred to as alpha-Glu, or simply aGlu, or simply Glu, due to the fact that it is the alpha carboxy group of the amino acid glutamic acid that can be used herein to other linker elements or for linking of lysine to the epsilon-amino group. .

Chem. 6 and Chem. The structure of 7 covers not only the D-type of Glu, but also the L-type.

The derivatives of the present invention may exist in different stereoisomeric forms, having the same molecular formula and sequence of the atoms to which they are attached, but differing only in the three-dimensional orientation of their atoms in space. The stereoisomerism of the exemplified derivatives of the present invention is indicated not only by structure but also by name using standard nomenclature in the experimental section. Unless otherwise stated, the invention relates to all stereoisomeric forms of the claimed derivatives.

The plasma concentration of the GLP-1 derivative of the present invention can be determined using any suitable method. For example, immunoassays such as LC-MS (liquid chromatography mass spectrometry) or RIA (radioimmunoassay), ELISA (enzyme immunoassay), and LOCI (luminescent oxygen channeling immunoassay) can be used. General protocols for suitable RIA and ELISA assays can be found in, for example, p. Found in 116-118. A preferred assay is the LOCI assay described in Examples 35, 39, and 40 herein.

Pharmacologically Acceptable Salt, amide, or ester

The derivatives and analogs of the present invention may be in the form of pharmaceutically acceptable salts, amides, or esters.

Salts are formed for example by a chemical reaction between a base and an acid, for example: NH ₃ + H ₂ S0 ₄ -> (NH ₄ ) ₂ S0 ₄ .

The salt may be a basic salt, an acid salt, or it may not be any (ie, a neutral salt). Basic salts give off hydroxide ions from water and acidic salts give off hydronium ions.

Salts of the derivatives of the present invention are formed with added cations or anions which react with anionic or cationic groups, respectively. These groups may be located in the peptide moiety and/or side chain of the derivatives of the invention.

Non-limiting examples of anionic groups of the derivatives of the invention include free carboxyl groups in the side chain, as well as in the peptide moiety in some cases. The peptide moiety often contains a free carboxyl group at the C-terminus, and it may also contain a free carboxy group at internal acidic amino acid residues such as Asp and Glu.

Non-limiting examples of cationic groups in the peptide moiety include free amino groups at the N-terminus, if present, as well as any free amino groups of internal basic amino acid residues such as His, Arg and Lys.

The esters of the derivatives of the invention can be formed, for example, by reaction of the free carboxyl group with an alcohol or phenol, which results in the replacement of at least one hydroxyl group by an alkoxy group or an aryloxy group.

The ester formation may include a free carboxyl group at the C-terminus of the peptide and/or any free carboxy group at the side chain.

The amides of the derivatives of the present invention can be formed, for example, by reaction with an amine of a free carboxyl group or a substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid.

Amide formation may include a free carboxyl group at the C-terminus of the peptide, a free carboxy group at the side chain, a free amino group at the N-terminus of the peptide, and/or any free or substituted amino group of the peptide at the peptide and/or side chain. have.

In certain embodiments, the peptide or derivative is in the form of a pharmaceutically acceptable salt. In another specific embodiment, the derivative is in the form of a pharmaceutically acceptable amide, preferably having an amide group at the C-terminus of the peptide. In a further specific embodiment, the peptide or derivative is in the form of a pharmaceutically acceptable ester.

Intermediate product

In a second aspect, the invention relates to an intermediate product.

One type of intermediate product of the invention compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 38Q; And/or (ii) a GLP-1 analog comprising a change in 39G; Or a pharmaceutically acceptable salt, amide, or ester thereof.

Other intermediate products of the invention in the form of a GLP-1 analog compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; (xxvi) 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q; Or (xxvii) analogs comprising, preferably having, amino acid changes of 8Aib, 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; Or a pharmaceutically acceptable salt, amide, or ester thereof.

Functional properties

In a first functional aspect, the derivatives of the invention have good efficacy. Also, or alternatively, in a second functional aspect, they have an extended pharmacokinetic profile. Also, or alternatively, in a third functional aspect, they have high oral bioavailability. Also, or alternatively, in a fourth functional aspect, their biophysical properties are improved.

Biological activity (efficacy)

According to a first functional aspect, the constituent GLP-1 peptide itself as well as the derivatives of the invention are biologically active or efficacious.

In certain embodiments, the efficacy and/or activity is the ability to stimulate the formation of cAMP in a cell line expressing a cloned human GLP-1 receptor, in vitro efficacy, i.e. performance in a functional GLP-1 receptor assay. Mention.

Stimulation of the formation of cAMP in media containing the human GLP-1 receptor can be accomplished using a stable transfected cell line such as BHK467-12A (tk-ts13), and/or added exogenously with, for example, cAMP formed endogenously. For the determination of cAMP, based on competition between biotin-labeled cAMPs, may be preferably determined using a functional receptor assay, wherein the assay cAMP is more preferably captured using a specific antibody, and/or an even more preferred assay. Is AlphaScreen cAMP Assay, most preferably one is described in Example 33.

The term, half of the maximum effective concentration (EC ₅₀ ), is a reference to the dose response curve and generally refers to the concentration that induces the intermediate response between the baseline and the maximum value. EC ₅₀ is used as a measure of the efficacy of the compound and represents the concentration at which 50% of its maximum effect is observed.

The in vitro efficacy of a derivative of the invention can be measured as _{described above, and by the measured EC 50 of the derivative.} The lower the EC ₅₀ , the better the efficacy.

Suitable media are: 50 mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl ₂ , 6H ₂ O; 150 mM NaCl; 0.01% Tween; 0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP; It has a composition of pH 7.4 (concentration for final analysis).

The EC ₅₀ of the inventive derivative is 3500 pM or below, preferably 3200 or below. The EC ₅₀ can even be below 1200 pM, preferably below 1000 pM, even more preferably below 500 pM, or most preferably below 200 pM.

In another specific embodiment of the first functional aspect, efficacy and/or activity refers to the ability to bind to the GLP-1 receptor with a low concentration of albumin. Binding to the GLP-1 receptor at low albumin concentrations should be as good as possible corresponding to _{low IC 50 values.} This can be measured as described in Example 35. _{The IC 50} (low albumin) of the inventive derivatives is 500 nM or below, and a lot below 100 nM, or even below 10 nM.

In another specific embodiment the derivatives of the invention have an in vivo efficacy that can be measured in any suitable animal model as well as in clinical trials, as known in the art.

Diabetic db/db mice are an example of a suitable animal model, and the blood glucose lowering effect is measured in such mice in vivo, for example as described in Example 36, or as described in Example 43 of WO09/030738. Can be.

In addition, or alternatively, the effect of food intake in vivo can be determined in a pharmacodynamic study of pigs, for example as described in Example 38.

Prolonged-receptor binding/low and high albumin

According to a second functional aspect, the derivatives of the invention are extended.

GLP-1 receptor binding

In certain embodiments, prolongation refers to the ability of a derivative of the invention to bind to the GLP-1 receptor in the presence of low and high concentrations of albumin, respectively, and can be measured as described in Example 34.

In general, binding to the GLP-1 receptor at low albumin concentrations should be as good as possible corresponding to _{low IC 50 values.} In one embodiment low albumin refers to 0.005% HSA. In another embodiment low albumin refers to 0.001% HSA.

_{The IC 50} value at high albumin concentration is a measure of the influence of albumin on the binding of the derivative to the GLP-1 receptor. As is known, GLP-1 derivatives also bind albumin. This is a generally desirable effect of extending their lifespan in plasma. Thus, the IC _{50 value for high albumin will generally be higher than the IC 50} value for low albumin, corresponding to reduced binding to the GLP-1 receptor, and by albumin binding competing for binding to the GLP-1 receptor. Is caused.

Thus, a high ratio (IC ₅₀ value (high albumin) / IC ₅₀ value (low albumin)) can be taken as an indication that the derivative binds well with albumin (may have a long half-life), and also itself is GLP- 1 Binds well with receptors (IC ₅₀ values (high albumin) are high, IC ₅₀ values (low albumin) are low). On the other hand, albumin binding may not always be desirable, or binding to albumin may become too strong. Thus, _{the preferred ranges for IC 50} (high albumin) / IC ₅₀ (low albumin), and low/low ratios are, depending on the intended use and the circumstances surrounding this use, and other compound properties of potential interest, from a compound to a compound Can be varied.

Suitable assays for measuring receptors that bind at high and low albumin concentrations are disclosed herein in Example 34. The compounds of the present invention have very good receptor binding affinity (IC ₅₀ ) in the presence of low albumin. _{The average IC 50} (low albumin) of the compound tested in Example 34 is 14 nM.

extension - Rat In vivo Half-life

According to a second functional aspect, the derivatives of the invention are extended. In certain embodiments, prolongation can suitably be measured as the in vivo half-life (T _{½) of a rat after intravenous administration.} The half-life of rats is at least 4 hours, which can be as high as 10 hours or more.

A suitable assay for determining the in vivo half-life of rats after intravenous administration is disclosed herein in Example 39.

Tools-Mini Pig's In vivo Half-life

According to a second functional aspect, the derivatives of the invention are extended. In addition, or alternatively, in certain embodiments, prolongation can be measured as _{the in vivo half-life (T ½) of a mini pig after intravenous administration.} The half-life is at least 12 hours, which can be at least 24 hours, at least 36 hours, at least 48 hours, or at least 60 hours, or even higher.

A suitable assay for determining the in vivo half-life of mini pigs after intravenous administration is disclosed in Example 37 herein.

Oral bioavailability

According to a third functional aspect, the derivatives of the invention have high oral bioavailability.

The oral bioavailability of commercial GLP-1 derivatives is very low. The oral bioavailability of GLP-1 derivatives under development for intravenous or subcutaneous administration is also low.

Therefore, there is a need for an improved oral bioavailability GLP-1 derivative in the present field. Such derivatives can be suitable candidates for oral administration as long as their efficacy is generally satisfactory, and/or their half-life is also generally satisfactory.

The inventors have surprisingly identified a new class of GLP-1 derivatives that have high oral bioavailability and at the same time have satisfactory efficacy and/or half-life.

In addition, or alternatively, these derivatives have surprisingly improved oral bioavailability and at the same time a high binding affinity to the GLP-1 receptor at low concentrations of albumin (i.e., low IC ₅₀ values).

These features are intended to obtain a low daily oral dose of the active pharmaceutical ingredient, which is desirable for a number of reasons including, for example, economics of manufacture, potential safety issues, as well as ease of administration issues and environmental issues. It is important.

In general, the term bioavailability refers to the portion of an administered dose of an active pharmaceutical ingredient (API), such as a derivative of the invention, that unchangedly reaches the systemic circulation. By definition, when an API is administered intravenously, its bioavailability is 100%. However, when it is administered via other routes (eg orally), its bioavailability decreases (due to degradation and/or incomplete absorption and first-pass metabolism). Knowledge of drug bioavailability is required when calculating dosages for non-intravenous routes of administration.

Absolute oral bioavailability compares the bioavailability of an API (area under the curve, or estimated as AUC) in the systemic circulation after oral administration to the bioavailability of the same API after intravenous administration. This is the portion of the API absorbed through non-intravenous administration compared to the corresponding intravenous administration of the same API. If different doses are used, the comparison should be dose normalized; Consequently, each AUC is corrected by dividing by the corresponding dose administered.

Plasma API concentration versus time plots are done after both oral and intravenous administration. Absolute bioavailability (F) is dose-corrected AUC-oral divided AUC-intravenous.

The derivatives of the present invention are higher than a) liraglutide, and/or b) semaglutide; Preferably at least 10% higher, more preferably at least 20% higher, even more preferably at least 30% higher, or most preferably at least 40% higher absolute oral bioavailability. Prior to testing for oral bioavailability, the derivatives of the invention may be suitably formulated as known in the art as oral preparations of insulin-secreting compounds, for example using any one or more preparations described in WO 2008/145728. I can.

An expected suitable test of oral bioavailability is described in Examples 35 and 40. According to these tests, after direct injection of the GLP-1 derivative into the intestinal lumen of the rat and/or after oral forced feeding of the rat, its concentration (exposure) in the plasma is determined, and subsequent exposure of the GLP-1 derivative to the plasma is determined. Is measured.

Biophysical Property

According to a fourth functional aspect, the derivatives of the invention have improved biophysical properties. These properties include, but are not limited to, physical stability and solubility. The improved biophysical properties can be the result of altered oligomer properties. Biophysical properties can be measured using standard biophysical methods of protein chemistry. The biophysical properties of the derivatives of the present invention can be suitably compared to the original GLP-1.

Further specific embodiments of the invention are described in the section headed "Specific Embodiments".

Manufacturing process

The preparation of peptides such as GLP-1(7-37) and GLP-1 analogs is well known in the art.

The GLP-1 portion (or fragment thereof) of a derivative of the invention, such as K ¹² , K ²⁷ -GLP-1 (7-37) or analogs or fragments thereof, can be used for example in classical peptide synthesis, e.g., t -Can be prepared by solid-phase peptide synthesis using Boc or Fmoc chemistry or other well-established techniques. For example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza Dorwald, "Organic Synthesis on Solide Phase", Wiley-VCH Verlag GmbH, 2000, and "Fmoc Solide Phase Peptide Synthesis"", Edited by WC Chan and PD White, Oxford University Press, 2000.

In addition, or alternatively, they can be prepared by recombinant methods, i.e., by culturing host cells capable of expressing the peptide in a suitable nutrient medium containing a DNA sequence encoding the analogue under conditions that permit expression of the peptide. . Non-limiting examples of host cells suitable for the expression of these peptides are: Escherichia coli , Saccharomyces cerevisiae ), as well as mammalian BHK or CHO cell lines.

Derivatives thereof of the invention comprising non-native amino acids and/or covalently attached N-terminal mono- or dipeptide mimetics can be prepared, for example, as described in the experimental section. Or, for example, Hodgson et al: "The ynthesis of Peptide and proteins containg non-natural amino acids", Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430; And WO 2009/083549 A1 entitled “Semi-recombinant preparation of GLP-1 analogues”.

Specific examples of methods of making numerous derivatives of the present invention are included in the experimental section.

Pharmaceutical composition

Pharmaceutical compositions comprising the derivatives of the present invention, or pharmaceutically acceptable salts, amides, or esters thereof, and pharmaceutically acceptable excipients may be prepared as known in the art.

The term “excipient” refers broadly to any ingredient other than the active therapeutic ingredient(s). The excipient may be an inert substance, a substance that is not active, and/or a substance that is not medicinally active.

Excipients may be provided for various purposes, for example as carriers, vehicles, diluents, tablet adjuvants, and/or to improve the administration and/or absorption of the active substance.

Formulations of active pharmaceutical ingredients with various excipients are known in the art. See, for example, Remington: The Science and Practice of Pharmacy (eg 19th edition (1995), and subsequent edits).

Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity modifiers, chelating agents, and stabilizers.

Examples of formulations include liquid formulations, ie aqueous formulations comprising water. Liquid formulations can be solutions or suspensions. Aqueous formulations typically comprise at least 50% w/w water, or at least 60%, 70%, 80%, or even at least 90% w/w water.

Alternatively, the pharmaceutical composition may be a solid formulation, for example, may be used as such, or may be a freeze-dried or spray-dried composition to which the physician or patient adds a solvent and/or diluent prior to use.

In aqueous formulations the pH can be anything between pH 3 and pH 10, for example about 7.0 to about 9.5; Or about 3.0 to about 7.0.

The pharmaceutical composition may comprise a buffer solution. Buffers include, for example, sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, bisine. , Tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, and mixtures thereof.

The pharmaceutical composition may contain a preservative. Preservatives are, for example, phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2 -Phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imideurea, chlorohexidine, sodium dihydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlor Lefenesine (3p-chlorphenoxypropane-1,2-diol), and mixtures thereof. The preservative may be present in a concentration of 0.1 mg/ml to 20 mg/ml.

The pharmaceutical composition may include an isotonic agent. Isotonic agents are, for example, salts (e.g. sodium chloride), sugars or sugar alcohols, amino acids (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), alditol (e.g. glycerol ( Glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol) polyethylene glycol (eg PEG400), and mixtures thereof. Any sugar, such as a monosaccharide, disaccharide, or polysaccharide, or for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha And a water-soluble glucan including beta HPCD, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na. Sugar alcohols are limited as C4-C8 hydrocarbons having at least one -OH group, and include, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcohol additive is mannitol.

The pharmaceutical composition may include a chelating agent. The chelating agent can be selected, for example, from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. The pharmaceutical composition may include a stabilizer. Stabilizers can be, for example, one or more oxidation inhibitors, aggregation inhibitors, surfactants, and/or one or more protease inhibitors. Non-limiting examples of these various types of stabilizers are disclosed below.

The term “aggregate formation” refers to physical interactions between polypeptide molecules, and oligomers capable of maintaining solubility are formed or large-looking aggregates are formed that precipitate out of solution. The formation of aggregates by the polypeptide during storage of the liquid pharmaceutical composition can adversely affect the biological activity of the polypeptide, resulting in loss of the therapeutic efficacy of the pharmaceutical composition. Moreover, aggregate formation can cause other problems such as clogging of tubing, membranes, or pumps when the polypeptide-containing pharmaceutical composition is administered using an infusion system.

The pharmaceutical composition may comprise an amount of amino acid base sufficient to reduce the formation of aggregates of the polypeptide during storage of the composition. The term “amino acid base” refers to one or more amino acids (eg, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), or analogs thereof. Certain amino acids can exist in its free base form or in its salt form. Certain stereoisomers of amino acid bases (ie, L, D, or mixtures thereof) may be present.

Methionine (or other sulfur amino acid or amino acid analog) can be added to inhibit the oxidation of methionine residues to methionine sulfoxides when the polypeptide acting as a therapeutic agent is a polypeptide comprising at least one methionine residue that is sensitive to such oxidation. Any stereoisomer of methionine (L or D) or combinations thereof may be used.

The pharmaceutical composition may include a stabilizer selected from the group of high molecular weight polymers or low molecular weight compounds. Stabilizers are, for example, polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivatives thereof (e.g. HPC, HPC-SL, HPC- L and HPMC), cyclodextrin, monothioglycerol, thioglycolic acid and sulfur-containing substances such as 2-methylthioethanol, and other salts (eg sodium chloride). Pharmaceutical compositions may include, but are not limited to, additional stabilizers such as methionine and EDTA that protect the polypeptide against methionine oxidation, and nonionic surfactants that protect the polypeptide against aggregation associated with freeze-thaw or mechanical shear. .

The pharmaceutical composition may comprise one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term “surfactant” refers to a water-soluble (hydrophilic) moiety, and any molecule or ion that contains a fat-soluble (lipophilic) moiety. The surfactant may be selected from the group consisting of, for example, anionic surfactants, cationic surfactants, nonionic surfactants, and/or amphoteric surfactants.

The pharmaceutical composition may comprise one or more protease inhibitors such as, for example, EDTA (ethylenediamine tetraacetic acid) and/or benzamidineHCl.

Additional optional components of the pharmaceutical composition include, for example, wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oily vehicles, proteins (e.g. human serum albumin, gelatin), and/or zwitterions (e.g. For example, amino acids such as betaine, taurine, arginine, glycine, lysine and histidine).

In addition, pharmaceutical compositions can be formulated using any one or more formulations described in, for example, WO 2008/145728, as known in the art as oral formulations of insulin-secreting compounds.

The administered dose may contain 0.01 mg-100 mg of the derivative, or 0.01-50 mg, or 0.01-20 mg, or 0.01-10 mg of the derivative.

The derivative can be administered in the form of a pharmaceutical composition. This can be done in some areas to the patient in need thereof, for example in local areas such as the skin or mucous membrane areas; At sites that bypass absorption, such as an artery, vein, or heart; And it can be administered at a site including absorption, such as intradermally, under the skin, intramuscularly, or in the abdomen.

The route of administration can be, for example, the tongue; Under the tongue; Cheeks; Oral; oral; Stomach; Intestines; Nasal cavity; Through the lungs, such as bronchioles, alveoli, or a combination thereof; Parenteral, epithelial; dermis; Transdermal; conjunctiva; Ureter; quality; rectal; And/or an eyeball. The composition may be an oral composition, and the route of administration is oral.

The composition may be in several dosage forms, for example as a solution; Suspension; Emulsion; Microemulsion; Multiple emulsions; Foam; Ointment; Paste; Plaster; Ointment; refine; Coated tablets; Chewing gum; Rinse; Capsules such as hard or soft gelatin capsules; suppository; Rectal capsule; Drop; Gel; Sprays; powder; Aerosol; Inhalants; eye drops; Ophthalmic ointment; Ophthalmic rinse; Jill Pessary; Vaginal rings; Vaginal ointment; Injection solution; In situ transformation solutions such as in situ gelling, curing, precipitation, and in situ crystallization; Infusion solution; Or can be administered as an implant. The composition may be an optionally coated tablet, capsule, or chewing gum.

The composition may be further compounded with a drug carrier or drug delivery system, for example to improve stability, bioavailability, and/or solubility. In certain embodiments, the composition may be attached to such systems through covalent, hydrophobic, and/or electrostatic interactions. The purpose of this unity may be, for example, to reduce adverse effects, achieve chronotherapy, and/or increase patient compliance.

The composition can also be used in the formulation of controlled, sustained, prolonged, retarded, and/or slow release drug delivery systems.

Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection by means of a syringe, optionally a pen-like syringe, or by an infusion pump.

The composition may be administered nasally in the form of a solution, suspension, or powder; Or it can be administered to the lungs in the form of a liquid or powder spray.

Transdermal administration may be of a further option, for example by needle-free injection, from a patch such as an iontophoretic patch, or via a transmucosal route, for example into the cheek.

The composition may be a stabilized formulation. The term "stabilized formulation" refers to a formulation having increased physical and/or chemical stability, preferably both. In general, the formulation should be stable during use and storage (depending on the recommended use and storage conditions) until the expiration date is reached.

The term “physical stability” refers to the tendency of a polypeptide to form biologically non-active and/or insoluble aggregates as a result of exposure to thermo-mechanical stress and/or interactions that destabilize interfaces and surfaces (such as hydrophobic surfaces). Mention. The physical stability of aqueous polypeptide preparations can be assessed by visual inspection and/or turbidity measurements after exposure to mechanical/physical stresses (eg agitation) for various time periods at different temperatures. Alternatively, physical stability can be assessed using spectroscopic agents or probes in the conformational state of the polypeptide, such as, for example, thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to a chemical (particularly covalent) change in the structure of a polypeptide that leads to the formation of a chemical degradation product that potentially has a reduced biological efficacy and/or increased immunogenic effect compared to an intact polypeptide. Chemical stability can be assessed by measuring the amount of chemical degradation products at various time points after exposure to different environmental conditions, for example by SEC-HPLC and/or RP-HPLC.

Treatment with the derivatives according to the invention may include, for example, antidiabetic agents, antiobesity agents, appetite regulators, antihypertensive agents, agents for the treatment and/or prevention of complications related to or related to diabetes and complications and disorders associated with or associated with obesity. It may also be combined with one or more additional pharmacologically active substances selected from agents for the treatment and/or prophylaxis of. Examples of these pharmacologically active substances are: insulin, sulfonylurea, biguanide, meglitinide, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, glucose production and/ Or compounds that modify lipid metabolism such as antihyperlipidemic agents such as inhibitors of liver enzymes involved in the stimulation of glycogenase, glucose uptake modulators, HMG CoA inhibitors (statins), gastric inhibitory polypeptides (GIP analogs), compounds that lower food intake, RXR agonists and agents that act on the ATP-dependent potassium channels of β-cells; Cholestyramine, cholestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; Β-blockers such as alpreolol, athenolol, thymolol, pindolol, propranolol and metoprolol, benazepril, captopril, enalapril, fosinopril, lisinopril, allatriopril, quinapril And ACE (angiotensin converting enzyme) inhibitors such as ramipril, nifedipine, felodipine, nicardipine, isradipine, nimodipine, calcium channel blockers such as diltiazem and verapamil, and doxazosin, urapidil, prazosin and terazosin Α-blockers such as; CART (cocaine amphetamine regulated transcription) agonist, NPY (neuropeptide Y) antagonist, PYY agonist, Y2 receptor agonist, Y4 receptor agonist, mixed Y2/Y4 receptor agonist, MC4 (melanocortin 4) agonist, orexin antagonist, TNF (tumor Necrosis factor) agonist, CRF (corticotropin releasing factor) agonist, CRF BP (corticotropin releasing factor binding protein) antagonist, urocortin agonist, β3 agonist, oxyntomodulin and analogs, MSH (melanin cell-stimulating hormone) ) Agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin reuptake inhibitors, serotonin and noradrenaline reuptake inhibitors, mixed serotonin and noradrenaline compounds, 5HT (serotonin) agonists, bombesin agonists, Galanine antagonists, growth hormones, growth hormone-releasing compounds, TRH (thyreotropin-releasing hormone) agonists, UCP 2 or 3 (binding inhibitory protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptine, doprexin) ), lipase/amylase inhibitors, RXR (retinoid X receptor) modulators, TR β agonists; Histamine H3 antagonists, gastric inhibitory polypeptide agonists or antagonists (GIP analogs), gastrin and gastrin analogs.

Treatment with the derivatives according to the invention can also be combined with surgery that affects glucose levels and/or lipid enhancement, such as gastric band implantation or gastric bypass grafting.

Pharmaceutical indications

In a third aspect, the invention also relates to a derivative of the invention for use as a medicament.

In certain embodiments, the derivatives of the invention can be used for the following medical treatments, all of which are preferably related in one way or differently to diabetes:

(i) prevention of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (pediatric diabetes mellitus), gestational diabetes, and/or a decrease in HbA1C, and/or cure;

(ii) Diabetic disease progression, such as progression of type 2 diabetes, delayed progression of impaired glucose tolerance (IGT) to insulin-demanding type 2 diabetes, and/or delayed progression of non-insulin-demanding type 2 diabetes to insulin-demanding type 2 diabetes Delay or prevention of;

(iii) improving β-cell function, such as reducing β-cell apoptosis, increasing β-cell function and/or β-cell mass, and/or restoring glucose sensitivity to β-cells;

(iv) prevention and/or treatment of cognitive impairment;

(v) prevention and/or treatment of eating disorders such as obesity, for example by reducing food intake, weight loss, suppressing appetite, inducing satiety; Treatment or prevention of anorexia, bulimia, and/or obesity caused by administration of antipsychotics or steroids; Decrease in gastric movement; And/or delayed gastric emptying;

(vi) neuropathy, including peripheral neuropathy; Nephropathy; Or prevention and/or treatment of diabetic complications such as retinopathy;

(vii) preventing and/or treating dyslipidemia, improving lipid parameters such as lowering total serum lipids; Lowering HDL; Lowering small and dense LDL; Lowering VLDL: lowering triglycerides; Lowering cholesterol; Increased HDL; Lowering plasma levels of lipoprotein a (Lp(a)) in humans; Inhibition of the development of apolipoprotein a(apo(a)) in vitro and/or in vivo;

(iix) syndrome X; Atherosclerosis; Myocardial infarction; Coronary heart disease; Stroke, cerebral ischemia; Early heart or early cardiovascular disease, such as an enlarged left ventricle; Coronary artery disease; Essential hypertension; Acute hypertensive urgency; Cardiomyopathy; Heart failure; Exercise load; Chronic heart failure; Arrhythmia; Heart rhythm disorder; swoon; Atherosclerosis; Mild chronic heart failure; angina pectoris; Heart bypass reperfusion; Intermittent claudication (obstructive atherosclerosis); Cardiac relaxation dysfunction; And/or the prevention and/or treatment of cardiovascular diseases such as systolic dysfunction;

(ix) inflammatory bowel syndrome; Small intestine syndrome, or Crohn's disease; Indigestion; And/or the prevention and/or treatment of gastrointestinal diseases such as gastric ulcers;

(x) the prevention and/or treatment of serious diseases, such as the treatment of severely ill patients, serious disease poly-nephropathy (CIPNP) patients, and/or potential CIPNP patients; Prevention of serious disease or development of CIPNP; Prevention, treatment and/or cure of Systemic Inflammatory Response Syndrome (SIRS) in a patient; And/or preventing or reducing the likelihood of a patient experiencing bacteremia, sepsis, and/or septic shock during hospitalization; And/or

(xi) Prevention and/or treatment of polycystic ovary syndrome (PCOS).

In certain embodiments, the indication is indication (i), (ii), and/or (iii); Or (i)-(iii) and (v)-(iix) such as signs (v), signs (vi), signs (vii), and/or signs (iix).

In another specific embodiment, the indication is (i). In a further specific embodiment the indication is (v). In a further specific embodiment the indication is (iix).

The following indications are: Type 2 diabetes, and/or obesity is particularly preferred.

certain Specific example

The following are specific embodiments of the present invention.

1.As a derivative of the GLP-1 analog, or a pharmaceutically acceptable salt, amide, or ester thereof,

Wherein the GLP-1 analog is the first K residue at the position corresponding to the 27 position of GLP-1 (7-37) (SEQ ID NO: 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;}

The derivatives are K ²⁷ and Comprising ^{two extension moieties attached to K T} via a first and a second linker, respectively,

Here, the first and second extended portions are the following Chem. 1 and Chem. Is selected from:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where x is an integer in the range 6-16 and y is an integer in the range 3-17;

The first and second linkers are described in Chem. Contains 5:

Chem. 5:

,

,

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

2. The derivative of embodiment 1, wherein T is an integer selected from the range 7-37 excluding 18 and 27.

3. The derivative of embodiment 1 or 2, wherein T is selected from any of the ranges 7-17, 19-26, and 28-37.

4. The derivative according to any of embodiments 1-3, wherein T is selected from the range 7-17.

5. The derivative according to any one of specific examples 1-4, wherein T is 12.

6. The derivative of any of embodiments 1-3, wherein T is selected from the range 19-26.

7. The derivative of any one of embodiments 1-3, and 6, wherein T is selected from the group consisting of 20, 22, and 24.

8. The derivative according to any one of embodiments 1-3 and 6-7, wherein T is 20.

9. The derivative according to any one of embodiments 1-3 and 6-7, wherein T is 22 or 24.

10. The derivative according to any one of embodiments 1-3, 6-7, and 9, wherein T is 22.

11. The derivative according to any one of embodiments 1-3, 6-7, and 9, wherein T is 24.

12. The derivative of any of embodiments 1-3, wherein T is selected from the range 28-37.

13. The derivative of any one of embodiments 1-3, and 12, wherein T is selected from the group consisting of 36 and 37.

14. The derivative of any of embodiments 1-3 and 12-13, wherein T is 36.

15. The derivative of any of embodiments 1-3 and 12, wherein T is 37.

16. The derivative according to any one of embodiments 1-16, wherein the position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1) is identified by handwriting and eyeballing.

17. The derivative according to any one of embodiments 1-16, wherein the position corresponding to the T position of GLP-1 (7-37) (SEQ ID NO: 1) is identified by handwriting and eye-balling.

18. The derivative of any one of embodiments 1-17, wherein the position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1) is identified by use of a standard protein or peptide alignment program.

19. The derivative of any one of embodiments 1-18, wherein the position corresponding to the T position of GLP-1 (7-37) (SEQ ID NO: 1) is identified by use of a standard protein or peptide alignment program.

20. The derivative of embodiment 19, wherein the alignment program is Needleman-Wunsch alignment.

21. The derivative of embodiment 19 or 20, wherein a default scoring matrix and a default identity matrix are used.

22. The derivative of any of embodiments 19-21, wherein the scoring matrix is BLOSUM62.

23. The derivative of any of embodiments 19-22, wherein the penalty for the first residue in the gap is -10 (minus 10).

24. The derivative of any of embodiments 19-23, wherein the penalty for additional residues in the gap is -0.5 (minus 0.5).

25. The derivative of any one of embodiments 1-24, wherein the analog does not comprise a K residue other than the first and second K residues.

26. The method of any of embodiments 1-25, wherein the extension moiety is Chem. 1 person derivative.

27. The derivative of any one of embodiments 1-26, wherein x is an even number.

28. The derivative of any of embodiments 1-27, wherein x is 12.

29. The method of any one of embodiments 1-28, wherein Chem. 1 is the following Chem. Derivatives represented by 1a:

Chem. 1a:

.

.

30. The method of any one of embodiments 1-25, wherein the extension moiety is Chem. 2-person derivative.

31. According to any one of embodiments 1-25, and 30, Chem. 2 is the following Chem. Derivatives represented by 2a:

Chem. 2a:

.

.

32. The derivative of any of embodiments 1-25 and 30-31, wherein y is an odd number.

33. The derivative of any of embodiments 1-25, and 30-32, wherein y is an integer in the range of 9-11.

34. The derivative of any of embodiments 1-25 and 30-33, wherein y is 9.

35. The derivative of any of embodiments 1-25 and 30-33, wherein y is 11.

36. According to any one of embodiments 1-25, and 30-35, Chem. 2 is the following Chem. 2b, or Chem. Derivatives represented by 2c:

Chem. 2b:

, 또는

, or

Chem. 2c:

.

.

37. According to any one of embodiments 1-25, and 30-35, Chem. 2 is Chem. Derivatives represented by 2b.

38. The method of any of embodiments 31-35, wherein Chem. 2a is the following Chem. 2b, or Chem. Derivatives represented by 2c:

Chem. 2b:

, 또는

, or

Chem. 2c:

.

.

39. The method of any of embodiments 31-35 and 38, wherein Chem. 2a is Chem. Derivatives represented by 2b.

40. The method of any one of embodiments 1-39, wherein Chem. 5 is a derivative which is a first linker element.

41. The derivative of any of embodiments 1-40, wherein k is 1.

42. The derivative of any of embodiments 1-41, wherein n is 1.

43. The method of any one of embodiments 1-42, wherein Chem. 5 is included m times, wherein m is an integer in the range of 1-10.

44. The derivative of embodiment 43, wherein m is 2.

45. According to embodiment 43 or 44, when m is not 1, Chem. A derivative whose five elements are interconnected through amide bond(s).

46. The derivative of any one of embodiments 1-45, wherein the linker further comprises a second linker element.

47. The derivative of embodiment 46, wherein the second linker element is Glu di-radical.

48. The method of embodiment 46 or 47, wherein the second linker element is Chem. 6, and/or Chem. Derivatives selected from 7:

Chem. 6:

, 및/또는

, And/or

Chem. 7:

.

.

49. The method of embodiment 48, wherein the second linker element is Chem. 6 phosphorus derivatives.

50. The derivative of any of embodiments 46-49, wherein the Glu di-radical is included p times, wherein p is an integer in the range of 1-2.

51. The derivative of embodiment 50, wherein p is 1.

52. The derivative of embodiment 50, wherein p is 2.

53. The derivative of any of embodiments 46-52, wherein the Glu di-radical is a radical of L-Glu.

54. The method of any one of embodiments 46-53, wherein one or more Glu di-radicals and one or more Chem. A derivative whose five elements are interconnected through amide bond(s).

55. The method of any one of embodiments 46-54, wherein the linker is Chem. Derivatives consisting of Glu di-radicals of 5 and p times.

56. The derivative of embodiment 55, wherein (m, p) is (2,2) or (2,1).

57. The derivative of embodiment 56, wherein (m, p) is (2,1).

58. According to any one of embodiments 55-57, Chem. 5-element and p-number of Glu di-radicals are interconnected through amide bonds.

59. The derivative of any of embodiments 1-58, wherein the linker and the extension moiety are interconnected through an amide bond.

60. The derivative of any of embodiments 1-59, wherein the linker and the GLP-1 analog are interconnected through an amide bond.

61. The derivative of any of embodiments 1-60, wherein the linker is attached to the epsilon-amino group of the first or second K residue.

62. The derivative of any of embodiments 1-61, wherein the linker has 5 to 41 C-atoms.

63. The derivative of any of embodiments 1-62, wherein the linker has 17 or 22 C-atoms.

64. The derivative of any of embodiments 1-63, wherein the linker has 17 C-atoms.

65. The derivative of any of embodiments 1-63, wherein the linker has 22 C-atoms.

66. The derivative of any of embodiments 1-65, wherein the linker has 4 to 28 hetero atoms.

67. The derivative of any of embodiments 1-66, wherein the linker has 12 or 16 hetero atoms.

68. The derivative of any of embodiments 1-67, wherein the linker has 12 heteroatoms.

69. The derivative of any of embodiments 1-67, wherein the linker has 16 heteroatoms.

70. The derivative of any of embodiments 66-69, wherein the hetero atom is an N, and/or an O-atom.

71. The derivative of any of embodiments 1-70, wherein the linker has 1 to 7 N-atoms.

72. The derivative of any of embodiments 1-71, wherein the linker has 3 or 4 N-atoms.

73. The derivative of any of embodiments 1-72, wherein the linker has 3 N-atoms.

74. The derivative of any of embodiments 1-72, wherein the linker has 4 N-atoms.

75. The derivative of any of embodiments 1-74, wherein the linker has 3 to 21 O-atoms.

76. The derivative of any of embodiments 1-75, wherein the linker has 9 or 12 O-atoms.

77. The derivative of any of embodiments 1-76, wherein the linker has 9 O-atoms.

78. The derivative of any of embodiments 1-76, wherein the linker has 12 O-atoms.

79. The method of any one of embodiments 1-78, wherein the linker is Chem. Chem. 6 and 2 Consisting of 5 and interconnected via an amide bond in the indicated sequence, the linker is linked from its *-NH end to the *-CO end of the extension part, and a GLP-1 analog at its *-CO end K ²⁷ or Derivatives linked to the epsilon amino group of K ^T.

80. The method of any one of embodiments 1-78, wherein the linker is Chem. Chem. 5 and 1 Consisting of 6 and interconnected via an amide bond at the indicated sequence, the linker is linked from its *-NH end to the *-CO end of the extension, and GLP-1 at its free *-CO end Analog of K ²⁷ or Derivatives linked to the epsilon amino group of K ^T.

81. The method of any one of embodiments 1-78, wherein the linker is Chem. Chem. 6 and 2 Consisting of 5 and interconnected via an amide bond in the indicated sequence, the linker is linked from its *-NH end to the *-CO end of the extension part, and a GLP-1 analog at its *-CO end K ²⁷ or Derivatives linked to the epsilon amino group of K ^T.

82. The method of any one of embodiments 1-78, wherein the linker is Chem. Chem. 6 and 2 5, and Chem. 6 and is interconnected via an amide bond in the indicated sequence, the linker being linked from its *-NH end to the *-CO end of the extension portion, and at its *-CO end the K ^{27 of the GLP-1 analog} or Derivatives linked to the epsilon amino group of K ^T.

83. The derivative of any of embodiments 1-82, wherein the two extending moieties are substantially the same.

84. The derivative of any one of embodiments 1-83, wherein the two extending portions are a) at least 80%, b) at least 85%, c) at least 90%, d) at least 95%, or e) at least 99% identical. .

85. The method of any one of embodiments 1-83, wherein the two extending portions comprise a) at least 0.5; b) at least 0.6; c) at least 0.7, d) at least 0.8; e) at least 0.9; Or f) a derivative having a similarity of at least 0.99.

86. The derivative of any of embodiments 1-85, wherein the two extending moieties have a similarity of 1.0.

87. The derivative of any of embodiments 1-86, wherein the two linkers are substantially the same.

88. The derivative of any of embodiments 1-87, wherein the two linkers have a similarity of at least 0.5.

89. The method of any one of embodiments 1-88, wherein the two linkers comprise a) at least 0.6; b) at least 0.7, c) at least 0.8; d) at least 0.9; Or e) a derivative having a similarity of at least 0.99.

90. The derivative of any of embodiments 1-89, wherein the two linkers have a similarity of 1.0.

91. The derivative of any of embodiments 1-90, wherein the two albumin binding agents, such as two side chains consisting of an extension moiety and a linker, are substantially the same.

92. The method of any one of embodiments 1-91, wherein the two albumin binders, such as two side chains consisting of an extension moiety and a linker, are a) at least 80%, b) at least 85%, c) at least 90%, d) at least 95%, or e) at least 99% identical derivatives.

93. The method of any one of embodiments 1-92, wherein the two albumin binders, such as two side chains consisting of an extension moiety and a linker, comprise a) at least 0.5; b) at least 0.6; c) at least 0.7, d) at least 0.8; e) at least 0.9; Or f) a derivative having a similarity of at least 0.99.

94. The derivative of any one of embodiments 1-92, wherein the two albumin binding agents, such as two side chains consisting of an extension moiety and a linker, have a similarity of 1.0.

95. The derivative of any of embodiments 83-94, wherein the two chemical structures being compared are represented as fingerprints.

96. The method of embodiment 95, wherein the fingerprint is a) an ECFP_6 fingerprint; b) Unit Y fingerprint; And/or c) a derivative that is an MDL fingerprint.

97. The derivative according to any of embodiments 95-96, wherein the Tanimoto coefficient is preferably used to calculate the similarity or identity of two fingerprints.

98. The derivative of any one of embodiments 1-97, wherein the number of amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1) is identified by handwriting and eyeballing.

99. The derivative of any one of embodiments 1-98, wherein the number of amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1) is identified by use of a standard protein or peptide alignment program.

100. The derivative of embodiment 99, wherein the alignment program is Needleman-Wunsch alignment.

101. The derivative of embodiment 99 or 100, wherein a default scoring matrix and a default identity matrix are used.

102. The derivative of any of embodiments 99-101, wherein the scoring matrix is BLOSUM62.

103. The derivative of any of embodiments 99-102, wherein the penalty for the first residue in the gap is -10 (minus 10).

104. The derivative of any of embodiments 99-103, wherein the penalty for additional residues in the gap is -0.5 (minus 0.5).

105. According to any one of embodiments 1-104, the amino acid change(s) is at the position of GLP-1(7-37) (SEQ ID NO: 1): 8, 12, 20, 22, 23, 24, 25 , 26, 27, 30, 31, 34, 35, 36, 37, 38, and a derivative appearing at one or more positions corresponding to 39.

106. According to any one of embodiments 1-105, the analog is changed: Aib ⁸ , K ¹² , K ²⁰ , E ²² or K ²² , E ²³ , K ²⁴ , V ²⁵ , R ²⁶ or At least one of H ²⁶ , K ²⁷ , E ³⁰ , H ³¹ , G ³⁴ or R ³⁴ or Q ³⁴ , Des ³⁵ , K ³⁶ or Des ³⁶ , K ³⁷ or Des ³⁷ , E ³⁸ or Q ³⁸ , and/or G ³⁹ Derivatives comprising a.

107. The method of any one of embodiments 1-106, wherein the second K moiety is K ¹² wherein the analog is ^{in addition to the change K 27} i) a ^{change selected from G 34} , R ³⁴ , and Q ³⁴ , and ii) R ²⁶ And Derivatives further comprising a change selected from H ^26.

108. The method of any one of embodiments 1-106, wherein the second K residue is K ²⁰ , wherein the analogue is ^{in addition to the change K 27} i) a ^{change selected from G 34} , R ³⁴ , and Q ³⁴ , and ii) R ²⁶ And Derivatives further comprising a change selected from H ^26.

109. The method of any one of embodiments 1-106, wherein the second K residue is K ²² , wherein the analogue is ^{in addition to the change K 27} i) a ^{change selected from G 34} , R ³⁴ , and Q ³⁴ , and ii) R ²⁶ And Derivatives further comprising a change selected from H ^26.

110. The method of any one of embodiments 1-106, wherein the second K moiety is K ²⁴ wherein the analog is ^{in addition to the change K 27} i) a ^{change selected from G 34} , R ³⁴ , and Q ³⁴ , and ii) R ²⁶ And Derivatives further comprising a change selected from H ^26.

111.The method of any one of embodiments 1-106, wherein the second K residue is K ³⁶ , wherein the analogue is ^{in addition to the change K 27} i) a ^{change selected from G 34} , R ³⁴ , and Q ³⁴ , and ii) R ²⁶ And Derivatives further comprising a change selected from H ^26.

112. The method of any one of embodiments 1-106, wherein the second K residue is K ³⁷ , wherein the analogue is ^{in addition to the change K 27} i) a ^{change selected from G 34} , R ³⁴ , and Q ³⁴ , and ii) R ²⁶ And Derivatives further comprising a change selected from H ^26.

113. According to any one of embodiments 1-112, the analog is changed: Aib ⁸ , E ²² , E ²³ , V ²⁵ , E ³⁰ , H ³¹ , Des ³⁵ , Des ³⁶ , Des ³⁷ , E ³⁸ or Q ³⁸ , And/or a derivative comprising at least one of ^{G 39.}

114. The derivative of any of embodiments 1-113, wherein the analog comprises Aib ⁸ .

115. The derivative of any of embodiments 1-114, wherein the analog comprises E ²² .

116. The derivative of any of embodiments 1-115, wherein the analog comprises E ²³ .

117. The derivative of any of embodiments 1-116, wherein the analog comprises V ²⁵ .

118. The derivative of any of embodiments 1-117, wherein the analog comprises E ³⁰ .

119. The derivative of any of embodiments 1-118, wherein the analog comprises H ³¹ .

120. The derivative of any of embodiments 1-119, wherein the analog comprises Des ³⁷ .

121. The derivative of embodiment 120, wherein the analog comprises Des ³⁶ .

122. The derivative of embodiment 121, wherein the analog comprises Des ³⁵ .

123. The derivative of any of embodiments 1-119, wherein the analog comprises E ³⁸ or Q ³⁸ .

124. The derivative of embodiment 123, wherein the analog comprises Q ³⁸ .

125. The derivative of embodiment 123, wherein the analog comprises E ³⁸ .

126. The derivative of any of embodiments 123-125, wherein the analog comprises G ^39.

127. The derivative of embodiment 122 of GLP-1(7-34) (amino acids 1-28 of SEQ ID NO: 1).

128. The derivative of embodiment 121 of GLP-1(7-35) (amino acids 1-29 of SEQ ID NO: 1).

129. The derivative of embodiment 120 of GLP-1(7-36) (amino acids 1-30 of SEQ ID NO: 1).

130. The derivative of any one of embodiments 1-119, of GLP-1(7-37) (amino acids 1-31 of SEQ ID NO: 1).

131. The derivative of any one of embodiments 123-125, of GLP-1 (7-38) (amino acids 1-31 of SEQ ID NO: 1, plus one amino acid residue added at the C-terminus).

132. The derivative of embodiment 126 of GLP-1 (7-39) (amino acids 1-31 of SEQ ID NO: 1, plus two amino acid residues added at the C-terminus).

133. The derivative of any of embodiments 1-132, wherein the analog has a maximum of 9 amino acid changes.

134. The derivative of any of embodiments 1-132, wherein the analogue has at most 8 amino acid changes.

135. The derivative of any of embodiments 1-132, wherein the analogue has at most 7 amino acid changes.

136. The derivative of any of embodiments 1-132, wherein the analogue has at most 6 amino acid changes.

137. The derivative of any of embodiments 1-132, wherein the analogue has at most 5 amino acid changes.

138. The derivative of any of embodiments 1-132, wherein the analogue has at most 4 amino acid changes.

139. The derivative of any of embodiments 1-132, wherein the analogue has at most 3 amino acid changes.

140. The derivative of any of embodiments 1-132, wherein the analogue has at most 2 amino acid changes.

141. The derivative of any of embodiments 1-132, wherein the analogue has at most one amino acid change.

142. The derivative of any of embodiments 1-140, wherein the analog has at least one amino acid change.

143. The derivative of any of embodiments 1-139, wherein the analogue has at least two amino acid changes.

144. The derivative of any of embodiments 1-138, wherein the analogue has at least 3 amino acid changes.

145. The derivative of any of embodiments 1-137, wherein the analogue has at least 4 amino acid changes.

146. The derivative of any of embodiments 1-136, wherein the analogue has at least 5 amino acid changes.

147. The derivative of any of embodiments 1-135, wherein the analogue has at least 6 amino acid changes.

148. The derivative of any of embodiments 1-134, wherein the analogue has at least 7 amino acid changes.

149. The derivative of any of embodiments 1-133, wherein the analogue has at least 8 amino acid changes.

150. The derivative of any of embodiments 1-132, wherein the analogue has at least 9 amino acid changes.

151. The derivative of any of embodiments 1-132, wherein the analogue has one amino acid change.

152. The derivative of any of embodiments 1-132, wherein the analog has two amino acid changes.

153. The derivative of any of embodiments 1-132, wherein the analog has 3 amino acid changes.

154. The derivative of any of embodiments 1-132, wherein the analogue has 4 amino acid changes.

155. The derivative of any of embodiments 1-132, wherein the analogue has 5 amino acid changes.

156. The derivative of any of embodiments 1-132, wherein the analog has 6 amino acid changes.

157. The derivative of any of embodiments 1-132, wherein the analogue has 7 amino acid changes.

158. The derivative of any of embodiments 1-132, wherein the analogue has 8 amino acid changes.

159. The derivative of any of embodiments 1-132, wherein the analogue has 9 amino acid changes.

160. The derivative of any of embodiments 1-132, wherein the analog has 10 amino acid changes.

161. The derivative of any of embodiments 1-160, wherein the change(s) are independently substitutions, additions, and/or deletions.

162. The derivative of any one of embodiments 1-161, wherein the analog comprises a GLP-1 analog of formula I:

Formula I: Xaa ₇ -Xaa ₈ -Glu-Gly-Thr-Xaa ₁₂ -Thr-Ser-Asp-Xaa ₁₆ -Ser-Xaa _{18 -Xaa 19} -Xaa ₂₀ -Glu-Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa ₂₆ -Lys-Phe-Ile-Xaa ₃₀ -Xaa ₃₁ -Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -Xaa ₃₇ -Xaa ₃₈ -Xaa ₃₉ , where

Xaa ₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N ^α -acetyl-histidine , N ^α -formyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaa ₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 -Aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;

Xaa ₁₂ is Lys or Phe;

Xaa ₁₆ is Val or Leu;

Xaa ₁₈ is Ser, Arg, Asn, Gin, or Glu;

Xaa ₁₉ is Tyr or Gin;

Xaa ₂₀ is Leu, Lys, or Met;

Xaa ₂₂ is Gly, Glu, Lys, or Aib;

Xaa ₂₃ is Gln, Glu, or Arg;

Xaa ₂₄ is Ala or Lys;

Xaa ₂₅ is Ala or Val;

Xaa ₂₆ is Val, His, or Arg;

Xaa ₃₀ is Ala, Glu, or Arg;

Xaa ₃₁ is Trp or His;

Xaa ₃₄ is Glu, Asn, Gly, Gln, or Arg;

Xaa ₃₅ is Gly, Aib, or absent;

Xaa ₃₆ is Arg, Gly, Lys, or absent;

Xaa ₃₇ is Gly, Ala, Glu, Pro, Lys, Arg, or absent;

Xaa ₃₈ is Ser, Gly, Ala, Glu, Gin, Pro, Arg, or absent;

Xaa ₃₉ is Gly or absent.

163. The derivative of embodiment 162, wherein the analog is a GLP-1 analog of formula I.

164. The derivative of embodiment 162 or 163, wherein the peptide of formula I is an analog of GLP-1(7-37) (SEQ ID NO: 1).

165. The derivative according to any one of specific examples 162-164, wherein if Xaa ₃₈ is absent, Xaa _{39 is} also absent.

166. The method of any one of embodiments 162-165, wherein if Xaa ₃₇ is absent, Xaa ₃₈ And a derivative that is also absent from _{Xaa 39.}

167. The derivative according to any one of embodiments 162-166, wherein when Xaa ₃₆ is absent, Xaa ₃₇ , Xaa ₃₈ , and Xaa _{39 are} also absent.

168. The derivative according to any one of embodiments 162-167, wherein Xaa ₃₅ is absent, and Xaa ₃₆ , Xaa ₃₇ , Xaa ₃₈ , and Xaa _{39 are} also absent.

169. The method of any of embodiments 162-168, wherein Xaa ₇ is His; Xaa ₈ is Ala or Aib; Xaa ₁₂ is Lys or Phe; Xaa ₁₆ is Val; Xaa ₁₈ is Ser; Xaa ₁₉ is Tyr; Xaa ₂₀ is Leu or Lys; Xaa ₂₂ is Glu, Gly or Lys; Xaa ₂₃ is Gln or Glu; Xaa ₂₄ is Ala or Lys; Xaa ₂₅ is Ala or Val; Xaa ₂₆ is His or Arg; Xaa ₃₀ is Ala or Glu; Xaa ₃₁ is Trp or His; Xaa ₃₄ is Gly, Gin, or Arg; Xaa ₃₅ is Gly or absent; Xaa ₃₆ is Arg, Lys, or absent; Xaa ₃₇ is Gly, Lys, or absent; Xaa ₃₈ is Glu or Gin; And Xaa ₃₉ is Gly or an absent derivative.

170. The derivative of any of embodiments 162-169, wherein Xaa ₇ is His.

171. The derivative of any of embodiments 162-170, wherein Xaa ₈ is Ala.

172. The derivative of any of embodiments 162-170, wherein Xaa ₈ is Aib.

173. The derivative of any of embodiments 162-172, wherein Xaa ₁₂ is Lys.

174. The derivative of any of embodiments 162-172, wherein Xaa ₁₂ is Phe.

175. The derivative of any of embodiments 162-174, wherein Xaa ₁₆ is Val.

176. The derivative of any of embodiments 162-175, wherein Xaa ₁₈ is Ser.

177. The derivative of any of embodiments 162-176, wherein Xaa ₁₉ is Tyr.

178. The derivative of any of embodiments 162-177, wherein Xaa ₂₀ is Leu.

179. The derivative of any of embodiments 162-177, wherein Xaa ₂₀ is Lys.

180. The derivative of any of embodiments 162-179, wherein Xaa ₂₂ is Glu.

181. The derivative of any of embodiments 162-179, wherein Xaa ₂₂ is Gly.

182. The derivative of any of embodiments 162-179, wherein Xaa ₂₂ is Lys.

183. The derivative of any of embodiments 162-182, wherein Xaa ₂₃ is Gin.

184. The derivative of any of embodiments 162-182, wherein Xaa ₂₃ is Glu.

185. The derivative of any of embodiments 162-184, wherein Xaa ₂₄ is Ala.

186. The derivative of any of embodiments 162-184, wherein Xaa ₂₄ is Lys.

187. The derivative of any of embodiments 162-186, wherein Xaa ₂₅ is Ala.

188. The derivative of any of embodiments 162-186, wherein Xaa ₂₅ is Val.

189. The derivative of any of embodiments 162-188, wherein Xaa ₂₆ is His.

190. The derivative of any of embodiments 162-188, wherein Xaa ₂₆ is Arg.

191. The derivative of any of embodiments 162-190, wherein Xaa ₃₀ is Ala.

192. The derivative of any of embodiments 162-190, wherein Xaa ₃₀ is Glu.

193. The derivative of any of embodiments 162-192, wherein Xaa ₃₁ is Trp.

194. The derivative of any of embodiments 162-192, wherein Xaa ₃₁ is His.

195. The derivative of any of embodiments 162-194, wherein Xaa ₃₄ is Gly.

196. The derivative of any of embodiments 162-194, wherein Xaa ₃₄ is Gin.

197. The derivative of any of embodiments 162-194, wherein Xaa ₃₄ is Arg.

198. The derivative of any of embodiments 162-197, wherein Xaa ₃₅ is Gly.

199. The derivative of any of embodiments 162-198, wherein Xaa ₃₅ is absent.

200. The derivative of any of embodiments 162-199, wherein Xaa ₃₆ is Arg.

201. The derivative of any of embodiments 162-199, wherein Xaa ₃₆ is Lys.

202. The derivative of any of embodiments 162-199, wherein Xaa ₃₆ is absent.

203. The derivative of any of embodiments 162-202, wherein Xaa ₃₇ is Gly.

204. The derivative of any of embodiments 162-202, wherein Xaa ₃₇ is Lys.

205. The derivative of any of embodiments 162-202, wherein Xaa ₃₇ is absent.

206. The derivative of any of embodiments 162-205, wherein Xaa ₃₈ is Glu.

207. The derivative of any of embodiments 162-205, wherein Xaa ₃₈ is Gin.

208. The derivative of any of embodiments 162-205, wherein Xaa ₃₈ is absent.

209. The derivative of any of embodiments 162-208, wherein Xaa ₃₉ is Gly.

210. The derivative of any of embodiments 162-208, wherein Xaa ₃₉ is absent.

211. The method of any of embodiments 1-210, wherein the analog is compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; (xxvi) 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q; Or (xxvii) a derivative comprising an amino acid change of 8Aib, 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G.

212. The derivative of embodiment 211, wherein the analog has a set of amino acid changes defined in any one of (i)-(xxvii).

213. Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66, Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72, Chem. 73, Chem. 74, Chem. 75, Chem. 76, Chem. 77, Chem. 78, Chem. 79, Chem. 80, and Chem. A compound selected from 81; Or a pharmaceutically acceptable salt, amide, or ester thereof.

214. A compound of embodiment 213, which is a compound according to any one of embodiments 1-212.

215. A compound characterized by its name and selected from the enumeration of the respective names of the compounds of Examples 1-32 herein, or a pharmaceutically acceptable salt, amide, or ester thereof.

216. The compound of embodiment 215 according to any one of embodiments 1-214.

217. The derivative according to any one of embodiments 1-216, which has GLP-1 activity.

218. The derivative of embodiment 217, wherein the GLP-1 activity refers to the ability to activate the human GLP-1 receptor.

219. The derivative of embodiment 217, wherein activation of the human GLP-1 receptor is measured by an in vitro assay.

220. The derivative of any of embodiments 217-219, wherein activation of the human GLP-1 receptor is measured as the efficacy of cAMP production.

221. The derivative of any one of embodiments 217-220 having an efficacy corresponding _{to the following EC 50:}

a) below 10000 pM, more preferably below 5000 pM, even more preferably below 4000 pM, or most preferably below 3000 pM;

b) below 2000 pM, preferably below 1500 pM, more preferably below 1200 pM, even more preferably below 1000 pM, or most preferably below 500 pM;

c) below 400 pM, preferably below 300 pM, more preferably below 200 pM, even more preferably below 150 pM, or most preferably below 100 pM; or

d) below 80 pM, preferably below 60 pM, more preferably below 40 pM, even more preferably below 30 pM, or most preferably below 20 pM.

222. The derivative of any of embodiments 217-221, wherein the efficacy is determined as _{EC 50} for a dose-response curve indicating the dose-dependent formation of cAMP in a medium containing human GLP-1 receptor.

223. The derivative of any of embodiments 219-222, which is a stable transfected cell line such as BHK467-12A (tk-ts13).

224. The derivative according to any one of embodiments 219-223, which is a functional receptor assay for the determination of cAMP.

225. The derivative of any of embodiments 219-224, wherein the assay is based on competition between endogenously formed cAMP and exogenously added biotin-labeled cAMP.

226. The derivative of any of embodiments 219-225, wherein the assay cAMP is captured using a specific antibody.

227. The derivative according to any of embodiments 219-226, which is an assay AlphaScreen cAMP Assay.

228. The derivative of any of embodiments 219-227, wherein the assay is described in Example 33.

229. According to any one of embodiments 217-228, the activation of the human GLP-1 receptor is measured as the ability to bind to the receptor in the presence of low albumin concentration, wherein the low albumin concentration is 0.005% HSA, or, preferably , A derivative that is 0.001% HSA.

230. According to any one of embodiments 217-229, the ratio [GLP-1 receptor binding affinity in the presence of 2.0% HSA (high albumin) (IC ₅₀ ), divided by 0.001% in the presence of HSA (low albumin). GLP-1 receptor binding affinity (IC ₅₀ )] is the following derivatives:

a) at least 1.0, more preferably at least 10, even more preferably at least 25, or most preferably at least 50;

b) at least 60, preferably at least 70, more preferably at least 80, even more preferably at least 90, or most preferably at least 100;

c) at least 125, preferably at least 150, more preferably at least 200, more preferably at least 250, even more preferably at least 400, or most preferably at least 500; or

d) at least 600, preferably at least 800, even more preferably at least 900, or most preferably at least 1000.

231. The derivative of any one of embodiments 217-230, wherein the GLP-1 receptor binding affinity (IC ₅₀ ) in the presence of 0.001% HSA (low albumin) is:

a) below 1000 nM, preferably below 750 nM, more preferably below 500 nM, or most preferably below 400 nM; or

b) below 300 nM, preferably below 250 nM, more preferably below 200 nM, or most preferably below 100 nM; or

c) below 50.0 nM, preferably below 15.0 nM, more preferably below 10.0 nM, even more preferably below 5.0 nM, or most preferably below 1.0 nM

d) below 0.80 nM, preferably below 0.60 nM, more preferably below 0.40 nM, even more preferably below 0.30 nM, or most preferably below 0.20 nM.

232. The derivative according to any one of embodiments 217-231, wherein the GLP-1 receptor binding affinity (IC ₅₀ ) in the presence of 2.0% HSA (high albumin) is

a) below 1000 nM, more preferably below 900 nM, or most preferably below 800 nM; or

b) below 500 nM, preferably below 400 nM, more preferably below 300 nM, even more preferably below 150 nM, or most preferably below 50.0 nM.

166. The derivative of any of embodiments 217-232, wherein the binding affinity for the GLP-1 receptor is determined by displacement of ^{125 I-GLP-1 from the receptor.}

233. The derivative of any of embodiments 217-232, wherein the SPA binding assay is used.

234. The derivative of any of embodiments 217-233, wherein the GLP-1 receptor is prepared using a stable, transfected cell line.

235. The derivative according to any of embodiments 217-234, wherein a hamster cell line, preferably a baby hamster kidney cell line such as BHK tk-ts13, is used.

236. The derivative of any of embodiments 229-235, wherein the IC ₅₀ value is determined as the concentration that displaces 50% of ^{125 I-GLP-1 from the receptor.}

237. The derivative according to any of embodiments 1-236, which has an oral bioavailability higher than semaglutide, preferably an absolute oral bioavailability.

238. The derivative of embodiment 237, wherein the oral bioavailability is measured in vivo in rats.

239. The derivative of any of embodiments 237-239, wherein the oral bioavailability is measured as exposure to plasma after direct injection into the intestinal lumen.

240. The plasma concentration of the derivative (pM), divided by the concentration of the injected solution (μM), measured 30 minutes after injection of the derivative solution in the jejunum of the rat according to any one of embodiments 237-239 (30 Dose-corrected exposure in minutes) is a) at least 39, b) at least 40; c) at least 60; d) at least 80; e) at least 100; f) at least 125; Or g) at least 150 derivatives.

241.The plasma concentration of the derivative (pM), divided by the concentration of the injected solution (μM) (at 30 minutes, measured 30 minutes after injection of the derivative solution into the rat jejunum). Dose-corrected exposure) is a) at least 160, b) at least 180, c) at least 200, or d) at least 250.

242. The derivative of any of embodiments 237-241, wherein the GLP-1 derivative is tested at a concentration of 1000 uM in admixture with 55 mg/ml sodium caprate.

243. The derivative of any of embodiments 237-242, wherein male Sprague Dawley rats are used.

244. The derivative of any of embodiments 237-243, wherein the rat has a body weight of about 240 g upon arrival.

245. The derivative of any of embodiments 237-244, wherein the rat is fasted for about 18 hours prior to the experiment.

246. The derivative according to any of embodiments 237-245, wherein the rat is under general anesthesia after fasting and the derivative is injected into the jejunum.

247. The derivative according to any one of embodiments 237-246, wherein the derivative is administered to the proximal portion (10 cm end to the duodenum), or the middle intestine (50 cm to the cecum) in the jejunum.

248. According to any one of embodiments 237-247, 100 μl of the derivative is injected into the jejunal lumen through a catheter with a syringe, then 200 μl of air is pushed into the jejunum lumen with another syringe and then connected to the catheter. Derivatives that are left to prevent reflux to the catheter.

249. The blood sample (200 ul) of any one of embodiments 237-248 is collected from the tail vein to the EDTA tube at the desired interval, such as at times 0, 10, 30, 60, 120 and 240 minutes, and 20 minutes Derivatives centrifuged for 5 minutes at 10000G at 4°C into the inside.

250. The derivative of any of embodiments 237-249, wherein the plasma (eg 75 ul) is frozen immediately after separation and maintained at -20°C until analyzed for the plasma concentration of the derivative.

251. The derivative of any of embodiments 237-250, wherein LOCI (luminescent oxygen channeling immunoassay) is used to analyze the plasma concentration of the derivative.

252. The derivative of any one of embodiments 1-251, wherein the derivative is effective for lowering blood glucose in vivo in db/db mice.

253. The derivative of any one of embodiments 1-252, wherein the derivative is effective for lowering body weight in vivo in db/db mice.

254. The derivative of embodiment 252 or 253, wherein db/db mice are treated subcutaneously with a suitable range of doses of the GLP-1 derivative, and blood glucose and/or body weight are measured at appropriate intervals.

255. In any one of embodiments 252-254, the dose of the GLP-1 derivative is 0.3 nmol/kg, 1.0 nmol/kg, 3.0 nmol/kg, 10 nmol/kg, 30 nmol/kg, and 100 nmol/kg. Where kg is a derivative referring to the weight of the mouse.

256. According to any one of embodiments 252-255, the control is treated subcutaneously with a vehicle, preferably a medium, wherein the GLP-1 derivative is for example composition: 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, Derivatives that dissolve to pH 7.4.

257. The blood glucose of any one of embodiments 252-256 is measured at time -½ hours (half hour prior to dosing (t=0)), and at times 1, 2, 4, and 8 hours, or And/or a mouse weighing derivative.

258. The derivative of any of embodiments 252-257, wherein the glucose concentration is measured using the glucose oxidase method.

259. The method of any one of embodiments 252-258,

(i) ED ₅₀ (body weight (BW)) is calculated as a dose that produces a half-maximal effect at 8 hours of delta (eg, reduction) BW after subcutaneous administration of the derivative; And/or

(ii) ED ₅₀ (blood glucose (BG)) is a dose that produces a half-maximal effect at 8 and/or 24 hours of AUC (area under the curve) delta (e.g., decrease) BG after subcutaneous administration of the derivative Derivatives calculated.

260. The derivative of any of embodiments 252-259, wherein a sigmoidal dose-response relationship is preferably present with a clear definition of maximal response.

261. The derivative of any one of embodiments 1-260, which has an extended profile of action than liraglutide.

262. The derivative of embodiment 261, wherein prolongation refers to the in vivo half-life of the relevant animal species.

263. The derivative of embodiment 261 or 262, wherein the animal is a) a db/db mouse, b) a rat, c) a pig, and/or d) a mini pig.

264. The derivative of embodiment 263, wherein the animal is a mini pig.

265. The derivative of any of embodiments 261-264, wherein the derivative is administered i) subcutaneously, and/or ii) intravenously.

266. The derivative of any of embodiments 1-265, wherein the derivative is administered intravenously.

267. The derivative according to any one of embodiments 1-266, wherein the final half-life (T _½ ) after intravenous administration in mini pigs is:

a) at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, or most preferably at least 60 hours;

b) at least 7 hours, preferably at least 16 hours, more preferably at least 24 hours, even more preferably at least 30 hours, or most preferably at least 40 hours;

c) at least 50 hours, preferably at least 60 hours, more preferably at least 70 hours, even more preferably at least 80 hours, or most preferably at least 90 hours.

268. The derivative of any of embodiments 264-267, wherein the minipig is a male Gottingen minipig.

269. The derivative according to embodiment 267 or 268, wherein the mini-pig is 7-14 months of age.

270. The derivative of any of embodiments 267-269, wherein the weight of the mini-pig is 16-35 kg.

271. The derivative according to any one of embodiments 267-270, wherein the mini pigs are each accommodated, preferably fed once or twice a day in the SDS mini pig diet.

272. The derivative of any of embodiments 267-271, wherein the derivative is administered intravenously after at least 2 weeks of acclimatization.

273. The derivative of any of embodiments 267-272, wherein the animal is fasted for about 18 hours before dosing and for at least 4 hours after dosing, and has free access to water for the entire period.

274. According to any one of embodiments 267-273, the GLP-1 derivative is dissolved in a concentration suitable for 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4, preferably 20-60 nmol/ml. Derivative.

275. The derivative of any of embodiments 267-274, wherein the intravenous injection of the derivative is given in a volume corresponding to 1-2 nmol/kg.

276. The derivative of any of embodiments 1-275, which results in reduced food intake in pigs.

277. The derivative according to embodiment 276, wherein the intake is preferably reduced relative to the vehicle-treated or untreated control.

278. The method of embodiment 276 or 277,

Food intake (0-24 hours) is

a) 90% or lower relative to the vehicle-treated control, b) preferably 80% or lower, c) more preferably 70% or lower, d) even more preferably 60% or lower, or e) most Preferably 50% or lower derivatives.

279. The derivative of any of embodiments 276-278, wherein the food intake (0-24 hours) refers to the first 24 hours after administration of the derivative or vehicle.

280. The derivative of any of embodiments 276-279, wherein the pig is a female Landrace Yorkshire Duroc (LYD) pig.

281. The derivative of any of embodiments 276-280, wherein the pig is 3 months of age.

282. The derivative of any of embodiments 276-281, wherein the pig has a weight of 30-35 kg.

283. The derivative of any of embodiments 276-282, wherein the animal is housed as a group for 1-2 weeks for adaptation.

284. The derivative of any one of embodiments 276-283, wherein the animal is placed in a separate fence from Monday morning to Friday afternoon for measurement of individual food intake during the experiment.

285. The derivative of any of embodiments 276-284, wherein the animal is fed freely with pig feed (eg Svinefoder, Antonio).

286. The derivative according to any of embodiments 276-285, wherein the food intake is monitored by recording the weight of the feed online every 15 minutes, preferably using the Mpigwin system.

287. The derivative of any of embodiments 276-286, which is administered at 0.3, 1.0, 3.0, 10, or 30 nmol/kg.

288. According to any one of embodiments 276-287, in a phosphate buffer (50 mM phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 8), preferably 12, 40, 120, 400, or 1200 nmol/ml Derivatives soluble in a concentration of.

289. The derivative of any one of embodiments 276-288, wherein the phosphate buffer solution serves as a vehicle.

290. According to any one of embodiments 276-289, the animal is administered as a single subcutaneous dose of the derivative on the morning of Day 1, or the vehicle (preferably in a dose volume of 0.025 ml/kg), and the food intake is dosed. Derivatives measured after 4 days.

_{291. The in vivo half-life of the rat according to any one of embodiments 1-290 (T ½} ) after intravenous administration a) at least 4 hours, b) at least 6 hours, c) at least 8 hours, or d) at least 10 hours. Derivatives with ).

_{292. The in vivo half-life of the rat according to any one of embodiments 1-291 (T ½} ) after intravenous administration a) at least 12 hours, b) at least 15 hours, c) at least 18 hours, or d) at least 20 hours. Derivatives with ).

_{293. The derivative of any one of embodiments 1-292, which has a rat in vivo half-life (T ½} ) at a) at least 24 hours, b) at least 26 hours, or c) at least 30 hours after intravenous administration.

294. The derivative of any of embodiments 291-294, wherein the rat is a male Sprague Dawley rat having a body weight of about 400 g.

294. The dose-corrected (i.e. divided by the dose of pmol of the injected derivative) plasma exposure curve (i.e. concentration in plasma in pM versus time) for any one of embodiments 238-294. Derivatives for which the AUC of is determined (i.e., results are expressed as (minutes × pM/pmol) or simply minutes/L).

295. For embodiment 294, the AUC of the dose-corrected plasma exposure curve is

a) at least 50, preferably at least 100, or more preferably at least 150 min/L;

b) at least 200, preferably at least 250, more preferably at least 300, or most preferably at least 320 min/L; or

c) a derivative which is at least 1.5 times, preferably at least 2 times, more preferably at least 3 times, or most preferably at least 4 times the corresponding AUC value for semaglutide.

296. The derivative according to any one of embodiments 1-295, wherein the oral bioavailability is exposure to plasma after oral forced feeding, which is measured in vivo in rats.

297.For embodiment 296, the AUC of the dose-corrected (i.e., divided by the dose of pmol of the injected derivative) plasma exposure curve (i.e., concentration in plasma as pM vs. time) from time 30 to 180 minutes is determined. Derivatives (i.e. results are expressed as (min × pM / pmol) or simply min/L)

298. For embodiment 297, the AUC of the dose-corrected plasma exposure curve is

a) at least 10, preferably at least 20, or more preferably at least 30 min/L;

b) at least 40, preferably at least 50, more preferably at least 60, or most preferably at least 70 min/L; or

c) a derivative which is at least 1.5 times, preferably at least 2 times, more preferably at least 3 times, or most preferably at least 4 times the corresponding AUC value for semaglutide.

299. A method according to any one of embodiments 294-298, GLP-1 derivative is sodium N of 250 mg / ml - about 1000 uM in a solution of [8-amino - (2-hydroxybenzoyl) caprylate (SNAC) Derivatives tested at a concentration of.

300. The derivative of any of embodiments 294-299, wherein male Sprague Dawley rats are used, preferably having a body weight of about 240 g upon arrival.

301. The derivative of any of embodiments 294-300, wherein the rat is fasted for about 18 hours prior to the experiment.

302. The derivative of any of embodiments 294-301, wherein the rat is subjected to general anesthesia after fasting and before injection of the derivative into the jejunum, or oral forced feeding, respectively.

303. The method of any one of embodiments 294-302, wherein for injection into the intestinal lumen, the derivative is in the proximal portion of the jejunum (10 cm distal to the duodenum) or the mid intestine (50 cm proximal to the cecum), preferably of the jejunum. Derivatives administered to the proximal part.

304. According to any one of embodiments 294-303, 100 μl of the derivative is injected into the jejunum lumen through a catheter with a 1 ml syringe, then 200 μl of air is pushed into the jejunum lumen with another syringe, and then the catheter. A derivative that is left to be connected to and prevents reflux to the catheter.

305. The blood sample (200 ul) of any one of embodiments 294-304 is collected from the tail vein to the EDTA tube at the desired interval, such as at times 0, 10, 30, 60, 120 and 240 minutes, and 20 minutes Derivatives centrifuged for 5 minutes at 10000G at 4°C into the inside.

306. The derivative of any of embodiments 294-305, wherein the plasma (eg 75 ul) is frozen immediately after separation and maintained at -20°C until analyzed for the plasma concentration of the derivative.

307. The derivative of any of embodiments 294-306, wherein LOCI (luminescent oxygen channeling immunoassay) is used to analyze the plasma concentration of the derivative.

308. Compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 38Q; And/or (ii) an intermediate product in the form of a GLP-1 analog comprising a change in 39G; Or a pharmaceutically acceptable salt, amide, or ester thereof.

309. The GLP-1 analog of embodiment 308 comprising (38E, 39G).

310. Compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; (xxvi) 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q; Or (xxvii) an intermediate product in the form of a GLP-1 analog comprising amino acid changes of 8Aib, 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; Or a pharmaceutically acceptable salt, amide, or ester thereof.

311. The GLP-analog of embodiment 310 having a set of amino acid changes defined by any of (i)-(xxvii).

312. The derivative according to any one of embodiments 1-307 for use as a medicament.

313. The treatment and/or prevention of all forms of diabetes and related diseases, such as dietary disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome according to any one of embodiments 1-307. For use in; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression, for use as such medicaments.

314. By administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-307-all forms of diabetes, such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome. And for the treatment and/or prevention of related diseases; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression.

The following are further specific embodiments of the invention:

1.As a derivative of the GLP-1 analog, or a pharmaceutically acceptable salt, amide, or ester thereof,

The analogue comprises a first K residue at a position corresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;}

The derivatives are K ²⁷ and Contains two albumin binding moieties attached to K ^{T, wherein}

The albumin binding moiety is described in Chem. 1 and Chem. It includes an extended portion selected from two:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where x is an integer ranging from 6-18 and y is an integer ranging from 3-17;

The extension part is Chem. When 1, the albumin binding moiety is the following formula Chem. It is a condition to further include a linker of 5:

Chem. 5:

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

2. As the derivative of Embodiment 1, or a pharmaceutically acceptable salt, amide, or ester thereof,

The GLP-1 analog has a first K residue at a position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;}

The derivatives are K ²⁷ and It contains two albumin binding moieties attached to ^{K T, wherein}

The albumin binding moiety is described in Chem. Includes 2, extension parts:

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where y is an integer in the range 3-17.

3. The derivative of any one of the preceding embodiments, wherein the albumin binding moiety further comprises a linker.

4. According to any one of the preceding embodiments, the linker is i) Glu di-radical; And/or ii) the following formula Chem. Derivatives comprising the linker of 5:

Chem. 5:

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

5. In any one of the previous embodiments, the Glu di-radical is the following Chem. 6, and/or Chem. 7, preferably Chem. Derivatives selected from 6:

Chem. 6:

Chem. 7:

.

.

6. As a derivative of any one of the preceding embodiments, or a pharmaceutically acceptable salt, amide, or ester thereof,

The GLP-1 analog has a first K residue at a position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;}

The derivatives are K ²⁷ and It contains two albumin binding moieties attached to ^{K T, wherein}

The albumin binding moiety is

i) the following formula Chem. 1, extension part:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

(Where x is an integer in the range 6-18); And

ii) the following formula Chem. 5, linker:

Chem. 5:

(Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5).

7. As a derivative of any one of the preceding embodiments, or a pharmaceutically acceptable salt, amide, or ester thereof,

The GLP-1 analog has a first K residue at a position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;}

Its derivatives are K ²⁷ and respectively It comprises two extension moieties attached to ^{K T through a linker, wherein}

The extension part is the following Chem. 1 and Chem. Is selected from:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where x is an integer ranging from 6-18 and y is an integer ranging from 3-17;

The linker is Chem. Contains 5:

Chem. 5:

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

8. The derivative of any of the preceding embodiments, wherein T is an integer selected from the range 7-37 excluding 18 and 27.

9. The derivative of any of the preceding embodiments, wherein T is selected from any of the ranges 7-17, 19-26, and 28-37.

10. The derivative of any of the preceding embodiments, wherein T is selected from the range 7-17.

11. The derivative of any of the preceding embodiments, wherein T is 12.

12. The derivative of any of the preceding embodiments, wherein T is selected from the range 19-26.

13. The derivative of any of the preceding embodiments, wherein T is selected from the group consisting of 20, 22, and 24.

14. The derivative of any of the preceding embodiments, wherein T is 20.

15. The derivative of any of the preceding embodiments, wherein T is 22 or 24.

16. The derivative of any of the preceding embodiments, wherein T is 22.

17. The derivative of any of the preceding embodiments, wherein T is 24.

18. The derivative of any of the preceding embodiments, wherein T is selected from the range 28-37.

19. The derivative of any of the preceding embodiments, wherein T is selected from the group consisting of 36 and 37.

20. The derivative of any of the preceding embodiments, wherein T is 36.

21. The derivative of any of the preceding embodiments, wherein T is 37.

22. The derivative according to any one of the preceding embodiments, wherein the position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1) is identified by handwriting and eye-balling.

23. The derivative according to any one of the preceding embodiments, wherein the position corresponding to the T position of GLP-1 (7-37) (SEQ ID NO: 1) is identified by handwriting and eyeballing.

24. The derivative of any one of the preceding embodiments, wherein the position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1) is identified by use of a standard protein or peptide alignment program.

25. The derivative of any one of the preceding embodiments, wherein the position corresponding to the T position of GLP-1 (7-37) (SEQ ID NO: 1) is identified by use of a standard protein or peptide alignment program.

26. The derivative of any of the preceding embodiments, wherein the alignment program is Needleman-Wunsch alignment.

27. The derivative of any of the previous embodiments, wherein a default scoring matrix and a default identity matrix are used.

28. The derivative of any of the previous embodiments, wherein the scoring matrix is BLOSUM62.

29. The derivative of any of the preceding embodiments, wherein the penalty for the first residue in the gap is -10 (minus 10).

30. The derivative according to any of the preceding embodiments, wherein the penalty for additional residues in the gap is -0.5 (minus 0.5).

31. The derivative of any of the preceding embodiments, wherein the analog does not comprise a K residue other than the first and second K residues.

32. As in any of the previous embodiments, the extension moiety is Chem. 1 person derivative.

33. The derivative of any of the preceding embodiments, wherein x is an even number.

34. The derivative of any of the preceding embodiments, wherein x is 12.

35. As in any of the previous embodiments, Chem. 1 is the following Chem. Derivatives represented by 1a:

Chem. 1a:

,

,

Where x is limited to any of the previous embodiments.

36. As in any of the previous embodiments, the extension moiety is Chem. 2, preferably the following Chem. Derivatives that are 2a:

Chem. 2a:

Where y is limited to any of the previous embodiments.

37. The derivative of any of the preceding embodiments, wherein y is an odd number.

38. The derivative of any of the preceding embodiments, wherein y is 9.

39. As in any of the previous embodiments, Chem. 2 is the following Chem. 2b, or Chem. 2c; Preferably Chem. Derivatives represented by 2b:

Chem. 2b:

Chem. 2c:

;

;

Where y is limited to any of the previous embodiments.

39a. In any of the previous embodiments, Chem. 2a is the following Chem. 2b, or Chem. 2c; Preferably Chem. Derivatives represented by 2b:

Chem. 2b:

Chem. 2c:

;

;

Where y is limited to any of the previous embodiments.

40. As in any of the previous embodiments, Chem. Derivatives comprising 5.

41. As in any of the previous embodiments, Chem. 5 is a derivative which is a first linker element.

42. The derivative of any of the preceding embodiments, wherein k is 1.

43. The derivative of any of the preceding embodiments, wherein n is 1.

44. As in any of the previous embodiments, Chem. 5 is included m times, where m is an integer in the range of 1-10.

45. The derivative of any of the preceding embodiments, wherein m is 2.

46. In any of the previous embodiments, when m is not 1, Chem. A derivative whose five elements are interconnected through amide bond(s).

47. The method of any of the preceding embodiments, wherein the linker comprises a second linker element; Preferably a Glu di-radical; More preferably, the following Chem. 6, and/or Chem. Selected from 7; Most preferably Chem. Derivatives further comprising 6:

Chem. 6:

Chem. 7:

.

.

48. The derivative of any of the preceding embodiments, wherein the Glu di-radical is included p times, wherein p is an integer in the range of 1-2.

49. The derivative of any of the preceding embodiments, wherein p is 1.

50. The derivative of any of the preceding embodiments, wherein p is 2.

51. The derivative according to any of the preceding embodiments, wherein the Glu di-radical is a radical of L-Glu.

52. The method of any of the preceding embodiments, wherein one or more Glu di-radicals and one or more Chem. A derivative whose five elements are interconnected through amide bond(s).

53. The method of any of the preceding embodiments, wherein the linker is at number m Chem. Derivatives consisting of 5 and p-time Glu di-radicals.

54. The derivative according to any of the preceding embodiments, wherein (m,p) is (2,2) or (2,1), preferably (2,1).

55. According to any of the previous embodiments, Chem. 5-element and p-number of Glu di-radicals are interconnected through amide bonds.

56. The derivative of any of the preceding embodiments, wherein the linker and the extension moiety are interconnected via an amide bond.

57. The derivative according to any of the preceding embodiments, wherein the linker and the GLP-1 analog are interconnected via an amide bond.

58. The derivative of any of the preceding embodiments, wherein the linker is attached to the epsilon-amino group of the first or second K moiety.

59. The method of any of the preceding embodiments, wherein the linker comprises 5 to 41 C-atoms; Derivatives preferably having 17 or 22 C-atoms.

60. The derivative of any of the preceding embodiments, wherein the linker has 17 C-atoms.

61. The derivative of any of the preceding embodiments, wherein the linker has 22 C-atoms.

62. The method of any of the preceding embodiments, wherein the linker comprises 4 to 28 heteroatoms; Derivatives preferably having 12 or 16 hetero atoms.

63. The derivative of any of the previous embodiments, wherein the linker has 12 heteroatoms.

64. The derivative of any of the preceding embodiments, wherein the linker has 16 heteroatoms.

65. The derivative of any of the preceding embodiments, wherein the hetero atom is an N, and/or O-atom.

66. The method of any of the preceding embodiments, wherein the linker comprises 1 to 7 N-atoms; Derivatives preferably having 3 or 4 N-atoms.

67. The derivative of any of the preceding embodiments, wherein the linker has 3 N-atoms.

68. The derivative of any of the preceding embodiments, wherein the linker has 4 N-atoms.

69. The method of any of the preceding embodiments, wherein the linker comprises 3 to 21 O-atoms; Derivatives preferably having 9 or 12 O-atoms.

70. The derivative of any of the preceding embodiments, wherein the linker has 9 O-atoms.

71. The derivative of any of the preceding embodiments, wherein the linker has 12 O-atoms.

72. According to any one of the preceding embodiments, the linker is interconnected via an amide bond in the indicated sequence, 2 Chem. Chem. 6 and 2 5, the linker is connected from its *-NH end to the *-CO end of the extension moiety, and at its *-CO end K ²⁷ of the GLP-1 analog or Derivatives linked to the epsilon amino group of K ^T.

73. The method of any of the preceding embodiments, wherein the linker is interconnected via an amide bond in the indicated sequence, 2 Chem. Chem. 5 and 1 6, wherein the linker is connected from its *-NH end to the *-CO end of the extension moiety, and at its free *-CO end K ²⁷ of the GLP-1 analog or Derivatives linked to the epsilon amino group of K ^T.

74. The method of any of the preceding embodiments, wherein the linker is interconnected via an amide bond in the indicated sequence, wherein Chem. Chem. 6 and 2 5, the linker is connected from its *-NH end to the *-CO end of the extension moiety, and at its *-CO end K ²⁷ of the GLP-1 analog or Derivatives linked to the epsilon amino group of K ^T.

75. The method of any of the preceding embodiments, wherein the linker is interconnected via an amide bond in the indicated sequence, wherein Chem. Chem. 6 and 2 5, and Chem. 6, wherein the linker is connected from its *-NH end to the *-CO end of the extension moiety, and at its *-CO end K ²⁷ of the GLP-1 analog or Derivatives linked to the epsilon amino group of K ^T.

76. The method of any of the preceding embodiments, wherein the two extending portions are substantially the same; For example at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical derivatives.

77. The method of any of the preceding embodiments, wherein the two extending portions have a similarity of at least 0.5; Preferably at least 0.6; More preferably at least 0.7, or at least 0.8; Even more preferably at least 0.9; Or most preferably a derivative having a similarity of at least 0.99, such as 1.0.

78. The method of any of the preceding embodiments, wherein the two linkers have a similarity of at least 0.5; Preferably at least 0.6; More preferably at least 0.7, or at least 0.8; Even more preferably at least 0.9; Or most preferably a derivative having a similarity of at least 0.99, such as 1.0.

79. The method of any of the preceding embodiments, wherein the two albumin binding agents, such as two side chains consisting of an extension moiety and a linker, are substantially the same; For example at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical derivatives.

80. The method of any of the preceding embodiments, wherein the two albumin binding agents, such as two side chains consisting of an extension moiety and a linker, have a similarity of at least 0.5; Preferably at least 0.6; More preferably at least 0.7, or at least 0.8; Even more preferably at least 0.9; Or most preferably a derivative having a similarity of at least 0.99, such as 1.0.

81. The method of any of the previous embodiments, wherein the two chemical structures being compared are: a) an ECFP_6 fingerprint; b) Unit Y fingerprint; And/or c) represent as a fingerprint, such as an MDL fingerprint; Here the Tanimoto coefficient for each a), b) and c) is preferably a derivative used to calculate the similarity or identity of two fingerprints.

82. The derivative according to any of the previous embodiments, wherein the number of amino acid changes compared to GLP-1(7-37) (SEQ ID NO: 1) is identified by handwriting and eyeballing.

83. The derivative of any of the previous embodiments, wherein the number of amino acid changes compared to GLP-1(7-37) (SEQ ID NO: 1) is identified by a standard protein or peptide alignment program.

84. The derivative of any of the previous embodiments, wherein the alignment program is Needleman-Wunsch alignment.

85. The derivative of any of the previous embodiments, wherein a default scoring matrix and a default identity matrix are used.

86. The derivative of any of the previous embodiments, wherein the scoring matrix is BLOSUM62.

87. The derivative of any of the preceding embodiments, wherein the penalty for the first residue in the gap is -10 (minus 10).

88. The derivative of any of the previous embodiments, wherein the penalty for additional residues in the gap is -0.5 (minus 0.5).

89. In any one of the previous embodiments, the amino acid change(s) is at position in GLP-1(7-37) (SEQ ID NO: 1): 8, 12, 20, 22, 23, 24, 25, A derivative that is one or more positions corresponding to 26, 27, 30, 31, 34, 35, 36, 37, 38, and 39.

90. In any of the previous embodiments, the analogue is changed: Aib ⁸ , K ¹² , K ²⁰ , E ²² or K ²² , E ²³ , K ²⁴ , V ²⁵ , R ²⁶ or H ²⁶ , K ²⁷ , E ³⁰ , H ³¹ , G ³⁴ or R ³⁴ or Q ³⁴ , Des ³⁵ , K ³⁶ or Des ³⁶ , K ³⁷ or Des ³⁷ , E ³⁸ or A derivative comprising at least one of Q ³⁸ and/or G ^39.

91.In any of the previous embodiments, the second K residue is K ¹² , wherein ^{in addition to the change K 27} the analogue is i) G ³⁴ and A derivative further comprising a change selected from Q ³⁴ , and ii) a change selected from R ²⁶ and H ^26.

92. In any of the previous embodiments, the second K residue is K ²⁰ , wherein ^{in addition to the change K 27} the analogue is i) G ³⁴ and A derivative further comprising a change selected from Q ³⁴ , and ii) a change selected from R ²⁶ and H ^26.

93. In any of the previous embodiments, the second K residue is K ²² , wherein ^{in addition to the change K 27} the analogue is i) G ³⁴ and A derivative further comprising a change selected from Q ³⁴ , and ii) a change selected from R ²⁶ and H ^26.

94. In any of the previous embodiments, the second K residue is K ²⁴ , wherein ^{in addition to the change K 27} the analogue is i) G ³⁴ and A derivative further comprising a change selected from Q ³⁴ , and ii) a change selected from R ²⁶ and H ^26.

95. In any of the previous embodiments, the second K residue is K ³⁶ , wherein ^{in addition to the change K 27} the analogue is i) G ³⁴ and A derivative further comprising a change selected from Q ³⁴ , and ii) a change selected from R ²⁶ and H ^26.

96. In any of the previous embodiments, the second K residue is K ³⁷ , wherein ^{in addition to the change K 27} the analogue is i) G ³⁴ and A derivative further comprising a change selected from Q ³⁴ , and ii) a change selected from R ²⁶ and H ^26.

97. In any of the previous embodiments, the analogue is changed: Aib ⁸ , E ²² , E ²³ , V ²⁵ , E ³⁰ , H ³¹ , Des ³⁵ , Des ³⁶ , Des ³⁷ , E ³⁸ or Q ³⁸ , and /Or a derivative comprising at least one of ^{G 39.}

98. The derivative of any of the previous embodiments, wherein the analog comprises Aib ^8.

99. The derivative of any of the preceding embodiments, wherein the analog comprises E ^22.

100. The derivative of any of the preceding embodiments, wherein the analog comprises E ^23.

101. The derivative of any of the preceding embodiments, wherein the analog comprises V ^25.

102. The derivative of any of the preceding embodiments, wherein the analog comprises E ^30.

103. The derivative of any of the preceding embodiments, wherein the analog comprises H ^31.

104. The derivative of any of the preceding embodiments, wherein the analog comprises Des ^35.

105. The derivative of any of the previous embodiments, wherein the analog comprises Des ^36.

106. The derivative of any of the preceding embodiments, wherein the analog comprises Des ^37.

107. According to any of the preceding embodiments, the analogue is E ³⁸ or Derivatives comprising Q ³⁸ , preferably Q ³⁸ , or more preferably E ^38.

108. The derivative of any of the preceding embodiments, wherein the analog comprises G ^39.

109. The derivative of any of the preceding embodiments, wherein the analog comprises Des ³⁵ , Des ³⁶ , and Des ³⁷ .

110. In any of the previous embodiments, the analog is Des ³⁶ And Des ³⁷ .

111. The derivative of any one of the preceding embodiments, of GLP-1(7-34) (amino acids 1-28 of SEQ ID NO: 1).

112. The derivative of any one of the preceding embodiments, of GLP-1(7-35) (amino acids 1-29 of SEQ ID NO: 1).

113. The derivative of any one of the preceding embodiments, of GLP-1(7-36) (amino acids 1-30 of SEQ ID NO: 1).

114. The derivative of any one of the preceding embodiments, of GLP-1(7-37) (amino acids 1-31 of SEQ ID NO: 1).

115. The derivative of any one of the preceding embodiments, of GLP-1 (7-38) (amino acids 1-31 of SEQ ID NO: 1, plus one amino acid residue added at the C-terminus).

116. The derivative of any one of the preceding embodiments, of GLP-1(7-39) (amino acids 1-31 of SEQ ID NO: 1, plus two amino acid residues added at the C-terminus).

117. The derivative of any of the preceding embodiments, wherein the analogue has at most 10 amino acid changes.

118. The derivative of any of the preceding embodiments, wherein the analog has a maximum of 9 amino acid changes.

119. The derivative according to any of the preceding embodiments, wherein the analogue has at most 8 amino acid changes.

120. The derivative of any of the previous embodiments, wherein the analog has a maximum of 7 amino acid changes.

121. The derivative of any of the preceding embodiments, wherein the analog has a maximum of 6 amino acid changes.

122. The derivative of any of the preceding embodiments, wherein the analog has a maximum of 5 amino acid changes.

123. The derivative of any of the preceding embodiments, wherein the analog has a maximum of 4 amino acid changes.

124. The derivative of any of the preceding embodiments, wherein the analog has a maximum of 3 amino acid changes.

125. The derivative of any of the preceding embodiments, wherein the analog has a maximum of 2 amino acid changes.

126. The derivative of any of the preceding embodiments, wherein the analog has at most one amino acid change.

127. The derivative of any of the preceding embodiments, wherein the analog has at least one amino acid change.

128. The derivative of any of the preceding embodiments, wherein the analog has at least two amino acid changes.

129. The derivative of any of the preceding embodiments, wherein the analog has at least 3 amino acid changes.

130. The derivative of any of the previous embodiments, wherein the analog has at least 4 amino acid changes.

131. The derivative of any of the preceding embodiments, wherein the analog has at least 5 amino acid changes.

132. The derivative of any of the preceding embodiments, wherein the analog has at least 6 amino acid changes.

133. The derivative of any of the preceding embodiments, wherein the analogue has at least 7 amino acid changes.

134. The derivative of any of the preceding embodiments, wherein the analog has at least 8 amino acid changes.

135. The derivative of any one of the preceding embodiments, wherein the analog has at least 9 amino acid changes.

136. The derivative of any of the preceding embodiments, wherein the analog has at least 10 amino acid changes.

137. The derivative of any of the preceding embodiments, wherein the analog has one amino acid change.

138. The derivative of any of the preceding embodiments, wherein the analog has a two amino acid change.

139. The derivative of any of the preceding embodiments, wherein the analog has a 3 amino acid change.

140. The derivative of any of the preceding embodiments, wherein the analog has a 4 amino acid change.

141. The derivative of any of the preceding embodiments, wherein the analog has a 5 amino acid change.

142. The derivative of any of the preceding embodiments, wherein the analog has 6 amino acid changes.

143. The derivative of any of the preceding embodiments, wherein the analog has a 7 amino acid change.

144. The derivative of any of the preceding embodiments, wherein the analog has an 8 amino acid change.

145. The derivative of any of the preceding embodiments, wherein the analog has a 9 amino acid change.

146. The derivative of any of the preceding embodiments, wherein the analog has a 10 amino acid change.

147. The derivative of any of the preceding embodiments, wherein the change(s) is independently a substitution, addition, and/or deletion.

148. In any of the previous embodiments, the analogue is

a) comprises a GLP-1 analog of formula I: And/or b) a derivative that is a GLP-1 analog of formula I:

Formula I: Xaa ₇ -Xaa ₈ -Glu-Gly-Thr-Xaa ₁₂ -Thr-Ser-Asp-Xaa ₁₆ -Ser-Xaa _{18 -Xaa 19} -Xaa ₂₀ -Glu-Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa ₂₆ -Lys-Phe-Ile-Xaa ₃₀ -Xaa ₃₁ -Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -Xaa ₃₇ -Xaa ₃₈ -Xaa ₃₉ , where

Xaa ₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N ^α -acetyl-histidine , N ^α -formyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine, or 4-pyridylalanine;

Xaa ₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 -Aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;

Xaa ₁₂ is Lys or Phe;

Xaa ₁₆ is Val or Leu;

Xaa ₁₈ is Ser, Arg, Asn, Gin, or Glu;

Xaa ₁₉ is Tyr or Gin;

Xaa ₂₀ is Leu, Lys, or Met;

Xaa ₂₂ is Gly, Glu, Lys, or Aib;

Xaa ₂₃ is Gln, Glu, or Arg;

Xaa ₂₄ is Ala or Lys;

Xaa ₂₅ is Ala or Val;

Xaa ₂₆ is Val, His, or Arg;

Xaa ₃₀ is Ala, Glu, or Arg;

Xaa ₃₁ is Trp or His;

Xaa ₃₄ is Glu, Asn, Gly, Gln, or Arg;

Xaa ₃₅ is Gly, Aib, or absent;

Xaa ₃₆ is Arg, Gly, Lys, or absent;

Xaa ₃₇ is Gly, Ala, Glu, Pro, Lys, Arg, or absent;

Xaa ₃₈ is Ser, Gly, Ala, Glu, Gin, Pro, Arg, or absent;

Xaa ₃₉ is Gly or absent.

149. The derivative of any one of the preceding embodiments, wherein the peptide of formula I is an analog of GLP-1(7-37) (SEQ ID NO: 1).

150. The derivative according to any of the preceding embodiments, wherein if Xaa ₃₈ is absent, then Xaa ₃₉ is also absent.

151. The method according to any of the preceding embodiments, if Xaa ₃₇ is absent, Xaa ₃₈ And a _{derivative in which Xaa 39} is also absent.

152. The derivative according to any of the preceding embodiments, wherein if Xaa ₃₆ is absent, then Xaa ₃₇ , Xaa ₃₈ , and Xaa ₃₉ are also absent.

153. The derivative of any of the preceding embodiments, wherein if Xaa ₃₅ is absent, then Xaa ₃₆ , Xaa ₃₇ , Xaa ₃₈ , and Xaa ₃₉ are also absent.

154. As in any of the previous embodiments, Xaa ₇ is His; Xaa ₈ is Ala or Aib; Xaa ₁₂ is Lys or Phe; Xaa ₁₆ is Val; Xaa ₁₈ is Ser; Xaa ₁₉ is Tyr; Xaa ₂₀ is Leu or Lys; Xaa ₂₂ is Glu, Gly or Lys; Xaa ₂₃ is Gln or Glu; Xaa ₂₄ is Ala or Lys; Xaa ₂₅ is Ala or Val; Xaa ₂₆ is His or Arg; Xaa ₃₀ is Ala or Glu; Xaa ₃₁ is Trp or His; Xaa ₃₄ is Gly, Gin, or Arg; Xaa ₃₅ is Gly or absent; Xaa ₃₆ is Arg, Lys, or absent; Xaa ₃₇ is Gly, Lys, or absent; Xaa ₃₈ is Glu or Gin; And Xaa ₃₉ is Gly or an absent derivative.

154a. The derivative of any of the preceding embodiments, wherein Xaa ₇ is His.

154b. The derivative of any of the preceding embodiments, wherein Xaa ₈ is Ala.

154b1. The derivative of any of the preceding embodiments, wherein Xaa ₈ is Aib.

154c. The derivative of any of the preceding embodiments, wherein Xaa ₁₂ is Lys.

154d. The derivative of any of the preceding embodiments, wherein Xaa ₁₂ is Phe.

154e. The derivative of any of the preceding embodiments, wherein Xaa ₁₆ is Val.

154f. The derivative of any of the preceding embodiments, wherein Xaa ₁₈ is Ser.

154g. The derivative of any of the preceding embodiments, wherein Xaa ₁₉ is Tyr.

154h. The derivative of any of the preceding embodiments, wherein Xaa ₂₀ is Leu.

154i. The derivative of any of the preceding embodiments, wherein Xaa ₂₀ is Lys.

154j. The derivative of any of the preceding embodiments, wherein Xaa ₂₂ is Glu.

154k. The derivative of any of the preceding embodiments, wherein Xaa ₂₂ is Gly.

154l. The derivative of any of the preceding embodiments, wherein Xaa ₂₂ is Lys.

154m. The derivative of any of the preceding embodiments, wherein Xaa ₂₃ is Gin.

154n. The derivative of any of the preceding embodiments, wherein Xaa ₂₃ is Glu.

154o. The derivative of any of the preceding embodiments, wherein Xaa ₂₄ is Ala.

154p. The derivative of any of the preceding embodiments, wherein Xaa ₂₄ is Lys.

154q. The derivative of any of the preceding embodiments, wherein Xaa ₂₅ is Ala.

154r. The derivative of any of the preceding embodiments, wherein Xaa ₂₅ is Val.

154s. The derivative of any of the preceding embodiments, wherein Xaa ₂₆ is His.

154t. The derivative of any of the preceding embodiments, wherein Xaa ₂₆ is Arg.

154u. The derivative of any of the preceding embodiments, wherein Xaa ₃₀ is Ala.

154v. The derivative of any of the preceding embodiments, wherein Xaa ₃₀ is Glu.

154x. The derivative of any of the preceding embodiments, wherein Xaa ₃₁ is Trp.

154y. The derivative of any of the preceding embodiments, wherein Xaa ₃₁ is His.

154z. The derivative of any of the preceding embodiments, wherein Xaa ₃₄ is Gly.

154aa. The derivative of any of the preceding embodiments, wherein Xaa ₃₄ is Gin.

154ab. The derivative of any of the preceding embodiments, wherein Xaa ₃₄ is Arg.

154ac. The derivative of any of the preceding embodiments, wherein Xaa ₃₅ is Gly.

154ad. The derivative of any of the preceding embodiments, wherein Xaa ₃₅ is absent.

154ae. The derivative of any of the preceding embodiments, wherein Xaa ₃₆ is Arg.

154af. The derivative of any of the preceding embodiments, wherein Xaa ₃₆ is Lys.

154ag. The derivative of any of the preceding embodiments, wherein Xaa ₃₆ is absent.

154ah. The derivative of any of the preceding embodiments, wherein Xaa ₃₇ is Gly.

154ai. The derivative of any of the preceding embodiments, wherein Xaa ₃₇ is Lys.

154aj. The derivative of any of the preceding embodiments, wherein Xaa ₃₇ is absent.

154ak. The derivative of any of the preceding embodiments, wherein Xaa ₃₈ is Glu.

154al. The derivative of any of the preceding embodiments, wherein Xaa ₃₈ is Gin.

154am. The derivative of any of the preceding embodiments, wherein Xaa ₃₈ is absent.

154an. The derivative of any of the preceding embodiments, wherein Xaa ₃₉ is Gly.

154ao. The derivative of any of the preceding embodiments, wherein Xaa ₃₉ is absent.

155. In any of the preceding embodiments, the analogue compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; Or (xxvi) 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q of amino acid changes containing, or preferably having a derivative.

156. Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66, Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72, Chem. 73, Chem. 74, Chem. 75, Chem. 76, Chem. 77, Chem. 78, and Chem. A compound selected from 79, preferably according to any one of the previous embodiments; Or a pharmaceutically acceptable salt, amide, or ester thereof.

157. A compound characterized by its name, selected from the enumeration of each name of the compound of Examples 1-30 herein, preferably according to any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof , Amides, or esters.

158. The derivative according to any of the preceding embodiments, having GLP-1 activity.

159. The derivative of any of the preceding embodiments, wherein the GLP-1 activity refers to the ability to activate the human GLP-1 receptor.

160. The derivative of any one of the preceding embodiments, wherein the activation of the human GLP-1 receptor is the efficacy of cAMP production, as measured by an in vitro assay.

161. The derivative according to any of the preceding embodiments, having an efficacy corresponding _{to the following EC 50:}

a) below 10000 pM, more preferably below 5000 pM, even more preferably below 4000 pM, or most preferably below 3000 pM;

b) below 2000 pM, preferably below 1500 pM, more preferably below 1200 pM, even more preferably below 1000 pM, or most preferably below 500 pM;

c) below 400 pM, preferably below 300 pM, more preferably below 200 pM, even more preferably below 150 pM, or most preferably below 100 pM; or

d) below 80 pM, preferably below 60 pM, more preferably below 40 pM, even more preferably below 30 pM, or most preferably below 20 pM.

_{162. In any of the preceding embodiments, the efficacy is measured as EC 50} to a dose-response curve indicating the dose-dependent formation of cAMP in a medium containing human GLP-1 receptor, preferably BHK467-12A Using a stable transfected cell line such as (tk-ts13), and/or using a functional receptor assay for the determination of cAMP, for example based on competition between endogenously formed cAMP and exogenously added biotin-labeled cAMP. Wherein the assay cAMP is more preferably captured using a specific antibody, and/or an even more preferred assay is the AlphaScreen cAMP Assay, most preferably one of the derivatives described in Example 31.

163. According to any of the previous embodiments, the ratio [GLP-1 receptor binding affinity in the presence of 2.0% HSA (high albumin) (IC ₅₀ ), divided by 0.001% HSA (low albumin) in the presence of GLP. -1 receptor binding affinity (IC ₅₀ )] is the following derivatives:

a) at least 1.0, more preferably at least 10, even more preferably at least 25, or most preferably at least 50;

b) at least 60, preferably at least 70, more preferably at least 80, even more preferably at least 90, or most preferably at least 100;

c) at least 125, preferably at least 150, more preferably at least 200, more preferably at least 250, even more preferably at least 400, or most preferably at least 500; or

d) at least 600, preferably at least 800, even more preferably at least 900, or most preferably at least 1000.

164. A derivative according to any of the preceding embodiments, wherein the GLP-1 receptor binding affinity (IC ₅₀ ) in the presence of 0.005% HSA (low albumin) is:

a) below 1000 nM, preferably below 750 nM, more preferably below 500 nM, or most preferably below 100 nM; or

b) below 50.0 nM, preferably below 15.0 nM, more preferably below 10.0 nM, even more preferably below 5.0 nM, or most preferably below 1.0 nM.

165. A derivative according to any of the preceding embodiments, wherein the GLP-1 receptor binding affinity (IC ₅₀ ) in the presence of 2.0% HSA (high albumin) is:

a) below 1100 nM, preferably below 1000 nM, more preferably below 900 nM, or most preferably below 600 nM; or

b) below 500 nM, preferably below 350 nM, more preferably below 200 nM, even more preferably below 100 nM, or most preferably below 50.0 nM.

166. The derivative according to any of the preceding embodiments, wherein the binding affinity for the GLP-1 receptor is ^{determined by displacement of 125} I-GLP-1 from the receptor, preferably using the SPA binding assay.

167. A derivative according to any of the preceding embodiments, wherein the GLP-1 receptor is prepared using a stable transfected cell line, preferably a hamster cell line, more preferably a baby hamster kidney cell line such as BHK tk-ts13.

168. The derivative of any of the preceding embodiments, wherein the IC ₅₀ value is determined as the concentration that displaces 50% of ^{125 I-GLP-1 from the receptor.}

169. The derivative according to any of the preceding embodiments, which has an oral bioavailability higher than semaglutide, preferably an absolute oral bioavailability.

170. The derivative of any of the preceding embodiments, wherein the oral bioavailability is the exposure to plasma after direct injection into the intestinal lumen, as measured in vivo in the rat.

171.The plasma concentration of the derivative (pM), divided by the concentration of the injected solution (μM) (dose at 30 minutes), measured 30 minutes after injection of the derivative solution into the jejunum of the rat, according to any of the previous embodiments. -Corrected exposure) is at least 39, or at least 40; Preferably at least 60; More preferably at least 80; More preferably at least 100; Even more preferably at least 125; Or most preferably at least 150 derivatives.

172. The plasma concentration of the derivative (pM), divided by the concentration of the injected solution (μM) (dose at 30 minutes), measured 30 minutes after injection of the derivative solution in the rat jejunum, according to any of the preceding embodiments. -Corrected exposure) is at least 160, preferably at least 180, more preferably at least 200, or most preferably at least 250.

173. The derivative of any of the preceding embodiments, wherein the GLP-1 derivative is tested at a concentration of 1000 uM in admixture with 55 mg/ml sodium caprate.

174. The derivative of any of the preceding embodiments, wherein male Sprague Dawley rats are used, preferably having a body weight of about 240 g upon arrival.

175. The derivative of any of the preceding embodiments, wherein the rat is fasted for about 18 hours prior to the experiment.

176. The derivative according to any of the preceding embodiments, wherein the rat is given general anesthesia after fasting and the derivative is injected into the jejunum.

177. The derivative according to any of the preceding embodiments, wherein the derivative is administered to the proximal portion of the jejunum (10 cm distal to the duodenum), or the mid intestine (50 cm proximal to the cecum).

178. In any of the previous embodiments, 100 μl of the derivative is injected into the jejunum lumen through a catheter with a syringe, followed by 200 μl of air being pushed into the jejunum lumen with another syringe, and then connected to the catheter. A derivative that is left behind and prevents reflux into the catheter.

179. In any of the preceding embodiments, a blood sample (200 ul) is collected from the tail vein into the EDTA tube at the desired interval, such as at times 0, 10, 30, 60, 120 and 240 minutes, and within 20 minutes. Derivatives centrifuged for 5 minutes at 10000G at 4℃.

180. The derivative according to any of the preceding embodiments, wherein the plasma (eg 75 ul) is frozen immediately after separation and maintained at -20° C. until analyzed for the plasma concentration of the derivative.

181. The derivative of any of the preceding embodiments, wherein LOCI (luminescent oxygen channeling immunoassay) is used to analyze the plasma concentration of the derivative.

182. The derivative according to any of the preceding embodiments, wherein the derivative is effective in lowering blood glucose in vivo in db/db mice.

183. The derivative according to any of the preceding embodiments, wherein the derivative is effective in lowering body weight in vivo in db/db mice.

184. The derivative of any of the preceding embodiments, wherein the db/db mice are treated subcutaneously with a suitable range of doses of the GLP-1 derivative and blood glucose and/or body weight are measured at appropriate intervals.

185. In any one of the previous embodiments, the dose of the GLP-1 derivative is 0.3 nmol/kg, 1.0 nmol/kg, 3.0 nmol/kg, 10 nmol/kg, 30 nmol/kg, and 100 nmol/kg. , Where kg is a derivative referring to the weight of the mouse.

186. In any of the preceding embodiments, the control is treated subcutaneously with a vehicle, preferably a medium, wherein the GLP-1 derivative is for example composition: 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH Derivatives soluble in 7.4.

187. In any of the preceding embodiments, at time -½ hour (half hour prior to dosing (t=0)), and at times 1, 2, 4, and 8 hours, blood glucose is measured, and /Or a derivative that weighs the mouse.

188. The derivative of any of the preceding embodiments, wherein the glucose concentration is measured using the glucose oxidase method.

189. In any of the previous embodiments,

(i) ED ₅₀ (body weight (BW)) is calculated as a dose that produces a half-maximal effect at 8 hours of delta (eg, reduction) BW after subcutaneous administration of the derivative; And/or

(ii) ED ₅₀ (blood glucose (BG)) is a dose that produces a half-maximal effect at 8 and/or 24 hours of AUC (area under the curve) delta (e.g., decrease) BG after subcutaneous administration of the derivative Derivatives calculated.

190. The derivative according to any of the preceding embodiments, wherein the sigmoidal dose-response relationship preferably exists with a clear definition of maximal response.

191. The derivative of any of the preceding embodiments, which has an extended profile of action than liraglutide.

192. In any of the preceding embodiments, prolongation refers to the in vivo half-life of a related animal species such as db/db mouse, rat, pig, and/or, preferably, mini-pig; Wherein the derivative is i) subcutaneous, and/or, ii) intravenous; Preferably ii) a derivative administered intravenously.

193. The derivative according to any of the preceding embodiments, wherein the final half-life (T _½ ) after intravenous administration in mini pigs is:

a) at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, or most preferably at least 60 hours;

b) at least 7 hours, preferably at least 16 hours, more preferably at least 24 hours, even more preferably at least 30 hours, or most preferably at least 40 hours;

c) at least 50 hours, preferably at least 60 hours, more preferably at least 70 hours, even more preferably at least 80 hours, or most preferably at least 90 hours.

194. The derivative according to any of the preceding embodiments, wherein the minipig is a male Gottingen minipig.

195. The derivative according to any of the preceding embodiments, wherein the minipig is 7-14 months of age, and preferably weighs 16-35 kg.

196. The derivative according to any of the preceding embodiments, wherein the mini-pigs are each housed, preferably fed once or twice a day in the SDS mini-pig diet.

197. The derivative of any of the preceding embodiments, wherein the derivative is administered intravenously after at least 2 weeks of acclimatization.

198. The derivative of any of the preceding embodiments, wherein the animal is fasted for about 18 hours before dosing and for at least 4 hours after dosing, and has free access to water for the entire period.

199. According to any of the preceding embodiments, the GLP-1 derivative is dissolved in a concentration suitable for 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4, preferably 20-60 nmol/ml. derivative.

200. The derivative according to any of the preceding embodiments, wherein the intravenous injection of the derivative is given in a volume corresponding to 1-2 nmol/kg.

201. The derivative of any one of the preceding embodiments, which increases glucose stimulated insulin secretion in mini pigs.

202. The derivative according to any of the preceding embodiments, wherein the minipig is a male Gottingen minipig.

203. The derivative according to any of the previous embodiments, wherein the mini-pig is 7-14 months of age.

204. The derivative according to any of the preceding embodiments, wherein the mini-pigs are housed in a single fence, preferably fed once or twice daily with SDS mini-pig feed.

205. The derivative of any of the preceding embodiments, wherein after a period of optionally dose escalation, a single dose is given intravenously or subcutaneously to the thin skin behind the ear.

206. The derivative of any of the preceding embodiments, wherein the animal is fasted for about 18 hours prior to dosing.

207. In any of the preceding embodiments, a baseline group and a number of derivative dose groups corresponding to 2-6 different plasma concentration levels are tested, wherein the baseline group is a) vehicle treated, or b) untreated derivative. .

208. The derivative of any of the preceding embodiments, wherein the plasma concentration level is 3000-80000 pM.

209. The derivative according to any of the preceding embodiments, wherein a 1 or 2 hour intravenous glucose tolerance test (IVGTT) is performed.

210. In any of the previous embodiments, 0.3 g/kg glucose is given intravenously over a period of 30 seconds and the blood sample is taken at the following time points (t=0 corresponds to glucose bolus): -10, -5 , 0, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 minutes.

211. The derivative of any one of the preceding embodiments, wherein the plasma concentration of the derivative, glucose, and insulin is measured.

212. A derivative according to any of the preceding embodiments, wherein the derivative concentration is measured at t=0 min, and optionally at the end of the test (t=60 min, or t=120 min).

213. The derivative of any of the preceding embodiments, wherein glucose is analyzed using the glucose oxidase method.

214. The derivative according to any of the preceding embodiments, wherein the area under the insulin curve (AUCinsulin) is calculated and used as a measure of insulin secretion.

215. According to any of the preceding embodiments, for at least one concentration thereof, the AUC insulin is higher than the baseline AUC insulin, preferably at least 110%, more preferably at least 120%, even more preferably At least 130% or most preferably at least 140% higher derivatives.

216. As a derivative of any of the preceding embodiments, resulting in a reduced food intake in pigs compared to a control (preferably vehicle-treated, or untreated);

Optionally, the food intake (0-24 hours) is 90% or lower, preferably 80% or lower, more preferably 70% or lower, even more preferably 60% or lower compared to the vehicle-treated control, or Most preferably can be 50% or lower;

Here, food intake (0-24 hours) refers to the first 24 hours after administration of the derivative or vehicle.

217. The derivative of any of the preceding embodiments, wherein the pig is a female Landrace Yorkshire Duroc (LYD) pig.

218. The derivative according to any of the preceding embodiments, wherein the pig has an age of 3 months, and preferably a weight of 30-35 kg.

219. The derivative according to any of the preceding embodiments, wherein the animal is housed as a group for 1-2 weeks for acclimatization.

220. The derivative according to any of the preceding embodiments, wherein the animal is placed in a separate fence from Monday morning to Friday afternoon for measurement of individual food intake during the experiment.

221. The derivative according to any of the preceding embodiments, wherein the animal is fed freely with pig feed (eg Svinefoder, Antonio).

222. The derivative according to any of the preceding embodiments, wherein the food intake is monitored by recording the weight of the feed online every 15 minutes, preferably using the Mpigwin system.

223. In any of the preceding embodiments, it is administered at 0.3, 1.0, 3.0, 10, or 30 nmol/kg, preferably a phosphate buffer (50 mM phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 8). ), more preferably at 12, 40, 120, 400, or 1200 nmol/ml.

224. The derivative according to any of the preceding embodiments, wherein the phosphate buffer acts as a vehicle.

225. In any of the preceding embodiments, the animal is administered as a single subcutaneous dose of the derivative on the morning of Day 1, or the vehicle (preferably in a dose volume of 0.025 ml/kg), and the food intake is after dosing. Derivatives measured over 4 days.

226. The in vivo half-life of the rat according to any one of the preceding embodiments, at least 4 hours, preferably at least 6 hours, even more preferably at least 8 hours, or most preferably at least 10 hours after intravenous administration. Derivatives with (T _{½ ).}

227. The in vivo half-life of the rat according to any of the preceding embodiments, at least 12 hours, preferably at least 15 hours, even more preferably at least 18 hours, or most preferably at least 20 hours after intravenous administration. Derivatives with (T _{½ ).}

228. The derivative according to any of the preceding embodiments, which has a rat in vivo half-life (T _½ ) at least 24 hours, preferably at least 26 hours, or most preferably at least 30 hours after intravenous administration.

229. The derivative of any of the preceding embodiments, wherein the rat is a male Sprague Dawley rat having a body weight of about 400 g.

230. Compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 38Q; And/or (ii) an intermediate product in the form of a GLP-1 analog comprising a change in 39G; Or a pharmaceutically acceptable salt, amide, or ester thereof.

231. The GLP-1 analog of embodiment 230 comprising (38E, 39G).

232. Compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; Or (xxvi) an intermediate product in the form of a GLP-1 analog, preferably having amino acid changes of 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q; Or a pharmaceutically acceptable salt, amide, or ester thereof.

233. A derivative according to any of the preceding embodiments for use as a medicament.

234. The treatment and/or prevention of all forms of diabetes and related diseases, such as dietary disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome according to any of the preceding embodiments. For use; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression.

235. By administering a pharmaceutically active amount of a derivative according to any of the preceding embodiments-all forms of diabetes, such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome, and For the treatment and/or prevention of related diseases; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression.

The following are further specific embodiments of the invention:

1.As a derivative of the GLP-1 analog, or a pharmaceutically acceptable salt, amide, or ester thereof,

The analogue comprises a first K residue at a position corresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); Comprises a second K residue at the position corresponding to the T position of GLP-1(7-37), wherein T is an integer ranging from 7-37 excluding 18 and 27; Up to 10 amino acids change compared to GLP-1 (7-37); Wherein the first K residue is ^{referred to as K 27} and the second K residue is referred to as ^{K T;}

The derivatives are K ²⁷ and It comprises two extension moieties attached to ^{K T through a linker, wherein}

The extension part is the following Chem. 1 and Chem. Is selected from:

Chem. 1: HOOC(CH ₂ ) _x -CO-*

Chem. 2: HOOC-C ₆ H ₄ -O-(CH ₂ ) _y -CO-*

Where x is an integer ranging from 6-18 and y is an integer ranging from 3-17; And

The linker is Chem. Contains 5:

Chem. 5:

Where k is an integer in the range of 1-5, and n is an integer in the range of 1-5.

2. In specific example 1, the linker is the following Chem. 6, and/or Chem. Derivatives further comprising a Glu di-radical selected from 7:

Chem. 6:

Chem. 7:

.

.

3. The derivative of any of the preceding embodiments, wherein the linker is attached to the epsilon-amino group of the first or second K moiety.

4. The derivative of any of the preceding embodiments, wherein T is 12, 20, 22, 24, 36, or 37.

5. The derivative according to any of the preceding embodiments, wherein the analog does not contain a K residue other than the first and second K residues.

6. The derivative of any of the preceding embodiments, wherein x is 12.

7. The derivative of any of the preceding embodiments, wherein y is 9.

8. The derivative of any of the preceding embodiments, wherein k is 1.

9. The derivative according to any of the preceding embodiments, wherein n is 1.

10. The derivative of any of the preceding embodiments, wherein the analog comprises a GLP-1 analog of formula I

Equation I:

Xaa ₇ -Xaa ₈ -Glu-Gly-Thr-Xaa ₁₂ -Thr-Ser-Asp-Xaa ₁₆ -Ser-Xaa _{18 -Xaa 19} -Xaa ₂₀ -Glu-Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa ₂₆ -Lys-Phe-Ile-Xaa ₃₀ -Xaa ₃₁ -Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -Xaa ₃₇ -Xaa ₃₈ -Xaa ₃₉ ,

here

Xaa ₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N ^α -acetyl-histidine , N ^α -formyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaa ₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 -Aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;

Xaa ₁₂ is Lys or Phe;

Xaa ₁₆ is Val or Leu;

Xaa ₁₈ is Ser, Arg, Asn, Gin, or Glu;

Xaa ₁₉ is Tyr or Gin;

Xaa ₂₀ is Leu, Lys, or Met;

Xaa ₂₂ is Gly, Glu, Lys, or Aib;

Xaa ₂₃ is Gln, Glu, or Arg;

Xaa ₂₄ is Ala or Lys;

Xaa ₂₅ is Ala or Val;

Xaa ₂₆ is Val, His, or Arg;

Xaa ₃₀ is Ala, Glu, or Arg;

Xaa ₃₁ is Trp or His;

Xaa ₃₄ is Glu, Asn, Gly, Gln, or Arg;

Xaa ₃₅ is Gly, Aib, or absent;

Xaa ₃₆ is Arg, Gly, Lys, or absent;

Xaa ₃₇ is Gly, Ala, Glu, Pro, Lys, Arg, or absent;

Xaa ₃₈ is Ser, Gly, Ala, Glu, Gin, Pro, Arg, or absent;

Xaa ₃₉ is Gly or absent.

11. Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66, Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72, Chem. 73, Chem. 74, Chem. 75, Chem. 76, Chem. 77, Chem. 78, and Chem. A compound according to any one of the previous embodiments selected from 79; Or a pharmaceutically acceptable salt, amide, or ester thereof.

12. Compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 38Q; And/or (ii) an intermediate product in the form of a GLP-1 analog comprising a change in 39G; Or a pharmaceutically acceptable salt, amide, or ester thereof.

13. The derivative according to any one of specific examples 1-11 for use as a medicament.

14. The treatment and/or prevention of all forms of diabetes and related diseases such as dietary disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome according to any one of embodiments 1-11. For use in; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression.

15. By administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-11-all forms of diabetes such as eating disorders, cardiovascular diseases, gastrointestinal diseases, diabetic complications, serious diseases, and/or polycystic ovary syndrome And for the treatment and/or prevention of related diseases; And/or for improving lipid parameters, for improving β-cell function, and/or for delaying or preventing diabetic disease progression.

Example

This experimental section begins with a list of abbreviations, followed by sections containing general methods for synthesizing and characterizing analogs and derivatives of the present invention. Then a number of examples relating to the preparation of specific GLP1 derivatives are followed, and at the end a number of examples relating to the activity and properties of these analogs and derivatives are included (the section is the subject of pharmacological methods).

The examples serve to illustrate the invention.

Abbreviation

The following abbreviations are used in alphabetical order below:

Aib: aminoisobutyric acid (α-aminoisobutyric acid)

API: active pharmaceutical ingredient

AUC: area under the curve

BG: blood glucose

BHK baby hamster kidney

BW: weight

Boc: t - butyloxycarbonyl

BSA: bovine serum albumin

Collidine: 2,4,6-trimethylpyridine

DCM: dichloromethane

Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl

DIC: diisopropylcarbodiimide

DIPEA: diisopropylethylamine

DMAP: 4-dimethylaminopyridine

DMEM: Dulbecco's Modified Eagle's Medium (DMEM)

EDTA: ethylenediaminetetraacetic acid

EGTA: ethylene glycol tetraacetic acid

FCS: Newborn baby serum

Fmoc: 9-fluorenylmethyloxycarbonyl

HATU: (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)

HBTU: (2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium hexafluorophosphate)

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol

HOAt: 1-hydroxy-7-azabenzotriazole

HOBt: 1-hydroxybenzotriazole

HPLC: high performance liquid chromatography

HSA: human serum albumin

IBMX: 3-isobutyl-1-methylxanthine

Imp: imidazopropionic acid (also referred to as des-amino histidine, DesH)

Intravenously intravenously

ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl

IVGTT: intravenous glucose tolerance test

LCMS: liquid chromatography mass spectrometry

LYD: Landrace Yorkshire Duroc

MALDI-MS: See MALDI-TOF MS

MALDI-TOF MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MeOH: methanol

Mmt: 4-methoxytrityl

Mtt: 4-methyltrityl

NMP: N-methyl pyrrolidone

OBz: benzoyl ester

OEG: 8-amino-3,6-dioxaoctanoic acid

OtBu: tert-butyl ester

PBS: phosphate buffered saline

PD: Pharmacodynamics

Pen/Strep: Penicillin/Streptomycin

PK: pharmacokinetic

RP: reversed phase

RP-HPLC: reverse phase high performance liquid chromatography

RT: room temperature

Rt: residence time

s.c.: subcutaneously

SD: standard deviation

SEC-HPLC: size exclusion high performance liquid chromatography

SEM: standard error of the mean

SPA: scintillation proximity

SPPS: solid phase peptide synthesis

tBu: tert-butyl

TFA: trifluoroacetic acid

TIS: triisopropylsilane

Tris: tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethyl-propane-1,3-diol

Trt: triphenylmethyl or trityl

Trx: Tranexamic acid

UPLC: ultra-high performance liquid chromatography

Method of manufacture

A. General method

This section is a method for synthesizing solid-phase peptides (the method of de-protecting amino acids, the method of cleaving the peptide from the resin, and the SPPS method, including its purification), as well as the method for detection and characterization of the resulting peptide (LCMS, MALDI, and UPLC method). In some cases, the solid phase synthesis of the peptide is di- with groups that can be cleaved under acidic conditions, such as but not limited to 2-Fmoc-oxy-4-methoxybenzyl, or 2,4,6-trimethoxybenzyl. It can be improved by the use of a di-peptide protected at the peptide amide bond. When serine or threonine is present in the peptide, pseudoproline di-peptide can be used (eg, commercially available from Novabiochem, see also W.R. Sampson (1999), J. Pep. Sci., 5, 403). The protected amino acid derivatives used were standard Fmoc-amino acids (supplied from eg Anaspec, IRIS, or Novabiochem). The N-terminal amino acid was Boc protected at the alpha amino group (eg Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides with His at the N-terminus). In the sequence, the epsilon amino group of lysine was protected with one of Mtt, Mmt, Dde, ivDde, or Boc, depending on the albumin binding moiety and the attachment pathway of the spacer. The albumin binding moiety and/or linker may be attached to the peptide by acylation of the resin binding peptide or by acylation in a solution of the unprotected peptide. In the case of the attachment of the albumin binding moiety and/or the linker to the protected peptidyl resin, the attachment is Fmoc-Oeg-OH (Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-Trx-OH (Fmoc -Tranexamic acid), Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester, nonadecanoic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy) benzoic acid tert-butyl ester and It may be modular, such as, but not limited to, suitably protected building blocks and using SPPS.

1. Synthesis of resin-binding peptide

SPPS Method B

SPPS Method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave-based Liberty peptide synthesizer (CEM Corp., North Carolina). A suitable resin is a preloaded, low-load Wang resin commercially available from Novabiochem (eg low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was carried out with 5% piperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solution (0.3 M in NMP at 3-10 fold molar excess) was added to the resin followed by an equal molar equivalent of DIC (0.75 M in NMP). For example, the following amounts of 0.3 M amino acid/HOAt solution can be used for the following scale reactions per coupling: scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. The coupling time and temperature were generally 5 minutes at 70 or 75° C. or less. Longer coupling times, eg 10 minutes, were used for larger scale reactions. Histidine amino acids were double-coupled at 50° C., or quadruple-coupled if the previous amino acid was steric hindrance (eg Aib). Arginine amino acids were coupled at RT for 25 minutes and then heated to 70 or 75° C. for 5 minutes. Some amino acids, such as but not limited to Aib, were "double-coupled", which after the first coupling (e.g., 5 minutes at 75° C.), the resin drained and more reagents (amino acids, HOAt and DIC ) Is added and the mixture is heated again (eg 5 minutes at 75°C). When chemical modification of the lysine side chain was desired, lysine was mixed as Lys (Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in pure (undiluted) hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. Chemical modification of lysine can be achieved by manual synthesis (see SPPS Method D) or one or more of the above-described Liberty peptide synthesizers, using suitably protected building blocks (see General Methods) and optionally including passive coupling. This was done by automated steps.

SPPS Method D

SPPS Method D refers to the synthesis of protected peptidyl resins using passive Fmoc chemistry. It is typically used for attachment of linkers and side chains to the peptide backbone. The following conditions were used on a 0.25 mmol synthesis scale. The coupling chemistry was DIC/HOAt/collidine in NMP at a 4-10 fold molar excess. The coupling conditions were 1-6 hours at room temperature. Fmoc-deprotection was performed with 20-25% piperidine in NMP (3 x 20 ml, 10 min each) followed by NMP washing (4 x 20 ml). Dde- or ivDde-deprotection was performed with 2% hydrazine in NMP (2 x 20 ml, 10 min each) followed by NMP washing (4 x 20 ml). Mtt- or Mmt-deprotection was performed with 2% TFA and 2-3% TIS in DCM (5×20 ml, 10 min each) followed by DCM (2×20 ml), 10% MeOH and 5% DIPEA in DCM (2 × 20 ml) and NMP (4 × 20 ml) were washed, or treated with pure hexafluroisopropanol (5 × 20 ml, 10 minutes each) and then washed as above. The albumin binding moiety and/or linker can be attached to the peptide by acylation of the resin binding peptide or by acylation in a solution of an unprotected peptide (see route described below). In the case of attachment of the albumin binding moiety and/or linker to the protected peptidyl resin, the attachment may be modular using SPPS and suitably protected building blocks (see General Methods).

Attachment to resin-binding peptide-Route I : Octadecanoic acid mono-(2,5-dioxo-pyrrolidin-1-yl) ester (Ebashi et al. EP511600, 4 molar equivalents to resin-binding peptide) and The same activated (active ester or symmetric anhydride) albumin binding moiety or linker was dissolved in NMP (25 ml), added to the resin, and shaken overnight at room temperature. The reaction mixture was filtered and the resin was washed extensively with NMP, DCM, 2-propanol, methanol and diethylether.

Attachment to resin-binding peptide-Route II : The albumin-binding portion was dissolved in NMP/DCM (1:1, 10 ml). Activation reagents such as HOBt (4 molar equivalents to resin) and DIC (4 molar equivalents to resin) were added and the solution was stirred for 15 minutes. The solution was added to the resin and DIPEA (4 molar equivalents to the resin) was added. The resin was shaken for 2 to 24 hours at room temperature. The resin was washed with NMP (2 x 20 ml), NMP/DCM (1: 1, 2 x 20 ml) and DCM (2 x 20 ml).

Attachment to Peptides in Solution-Route III : Activated (active ester or symmetric anhydride such as octadecanoic acid mono-(2,5-dioxo-pyrrolidin-1-yl) ester (Ebashi et al. EP511600)) ) 1-1.5 molar equivalents relative to the albumin binding moiety or linker peptide are dissolved in an organic solvent such as acetonitrile, THF, DMF, DMSO or a mixture of water/organic solvents (1-2 ml) and with 10 molar equivalents of DIPEA It was added to a solution of the peptide in water (10-20 ml). In the case of protecting groups at albumin binding moieties such as tert-butyl, the reaction mixture was freeze-dried overnight and then the isolated crude peptide was deprotected. Deprotection in the case of the tert -butyl protecting group was carried out by dissolving the peptide in a mixture of trifluoroacetic acid, water and triisopropylsilane (90:5:5). After 30 minutes the mixture was evaporated in vacuo and the crude peptide was purified by preparative HPLC described later.

SPPS Method E

SPPS Method E refers to peptide synthesis by Fmoc chemistry on Prelude Solid Phase Peptide Synthesiser from Protein Technologies (Tucson, AZ 85714 U.S.A.). A suitable resin is a preloaded, low-load Wang resin commercially available from Novabiochem (eg low load fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was performed for 2 x 10 minutes with 25% piperidine in NMP. The coupling chemistry was DIC/HOAt/collidine in NMP. Amino acid/HOAt solution (0.3 M in NMP at 3-10 fold molar excess) was added to the resin followed by equal molar equivalents of DIC (3 M in NMP) and collidine (3 M in NMP). For example, the following amounts of 0.3 M amino acid/HOAt solution can be used for the following scale reactions per coupling: scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml. The coupling time and temperature were generally 5 minutes at 70 or 75° C. or less. The coupling time was typically 60 minutes. Certain amino acids including, but not limited to, arginine, Aib or histidine were "double-coupled", which, after the first coupling (e.g. 60 minutes), the resin was drained and more reagents (amino acids, HOAt, DIC , And collidine) are added and the mixture is allowed to react again (e.g. 60 minutes). Fmoc-OEG-OH, Fmoc-Trx-OH, Fmoc-Glu-OtBu, octadecanoic acid mono-tert-butyl ester, nonadecanoic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy) Certain amino acid and fatty acid derivatives, including but not limited to benzoic acid tert-butyl ester, were coupled for a delayed time, for example 6 hours. When chemical modification of the lysine side chain was desired, lysine was mixed as Lys (Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in hexafluoroisopropanol/DCM (75:25) for 3 x 10 minutes followed by washing with DCM, 20% piperidine and NMP. Chemical modification of lysine was performed either by manual synthesis (see SPPS Method D) or by one or more automated steps (see General Methods) in the Prelude peptide synthesizer described above using suitably protected building blocks.

2. Cleavage of Peptides from Resin and Purification

After synthesis, the resin was washed with DCM, and the peptide was digested from the resin by 2-3 hours treatment with TFA/TIS/water (95/2.5/2.5 or 92.5/5/2.5) and then precipitated with diethyl ether. The peptide was dissolved in a suitable solvent (eg, 30% acetic acid) and purified by standard RP-HPLC on a C18, 5 μM column using acetonitrile/water/TFA. Fractions were analyzed by a combination of UPLC, MALDI and LCMS methods, and appropriate fractions were pooled and lyophilized.

3. Method of detection and characterization

LCMS Way

LCMS Method 1( LCMS1 )

An Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was used to determine the mass of the sample after elution from an Agilent 1200 series HPLC system. The de-convolution of the protein spectrum was calculated with Agilent's protein identification software.

Eluent:

A: 0.1% trifluoro acetic acid in water

B: 0.1% trifluoro acetic acid in acetonitrile

Column: Zorbax 5u, 300SB-C3, 4.8×50mm

Gradient: 25%-95% acetonitrile over 15 minutes

LCMS Method 2( LCMS2 )

A Perkin Elmer Sciex API 3000 mass spectrometer was used to determine the mass of the sample after elution from a Perkin Elmer series 200 HPLC system.

Eluent:

A: 0.05% trifluoro acetic acid in water

B: 0.05% trifluoro acetic acid in acetonitrile

Column: Waters Xterra MS C-18 × 3 mm inner diameter 5 μM

Gradient: 5%-90% acetonitrile over 7.5 min at 1.5 ml/min

LCMS Method 3( LCMS3 )

A Waters Micromass ZQ mass spectrometer was used to determine the mass of the sample after elution from the Waters Alliance HT HPLC system.

Eluent:

A: 0.1% trifluoro acetic acid in water

B: 0.1% trifluoro acetic acid in acetonitrile

Column: Phenomenex, Jupiter C4 50 × 4.60 mm inner diameter 5 μM

Gradient: 10%-90% B over 7.5 min at 1.0 ml/min

LCMS Method 4( LCMS4 )

LCMS4 was performed in a setup consisting of a Waters Acquity UPLC system and an LCT Premier XE mass spectrometer from Micromass. The UPLC pump was connected to two eluent reservoirs containing:

A: 0.1% formic acid in water

B: 0.1% formic acid in acetonitrile

The analysis was performed at RT by injecting an appropriate volume of sample (preferably 2-10 μl) onto the eluted column with a gradient of A and B.

UPLC conditions, detector settings and mass spectrometer settings:

Column: Waters Acquity UPLC BEH, C-18, 1.7 μM, 2.1 mm × 50 mm

Gradient: Linear 5%-95% acetonitrile for 4.0 min at 0.4 ml/min (alternatively 8.0 min)

Detection: 214 nm (analog output from TUV (Tunable UV detector))

MS ionization method: API-ES

Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu

UPLC And HPLC Way

Method 05_B5_1

UPLC (Method 05_B5_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na ₂ SO ₄ , 0.04 MH ₃ PO ₄ , 10% CH ₃ CN (pH 3.5)

B: 70% CH ₃ CN, 30% H ₂ O

Linear gradient: 60% A, 40% B to 30% A, 70% B were used over 8 minutes at a flow rate of 0.40 ml/min.

Method 05_B7_1

UPLC (Method 05_B7_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na ₂ SO ₄ , 0.04 MH ₃ PO ₄ , 10% CH ₃ CN (pH 3.5)

B: 70% CH ₃ CN, 30% H ₂ O

Linear gradient: 80% A, 20% B to 40% A, 60% B were used over 8 minutes at a flow rate of 0.40 ml/min.

Method 04_A2_1

UPLC (Method 04_A2_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 90% H ₂ O, 10% CH ₃ CN, 0.25 M ammonium bicarbonate

B: 70% CH ₃ CN, 30% H ₂ O

Linear gradient: 90% A, 10% B to 60% A, 40% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 04_A3_1

UPLC (Method 04_A3_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 90% H ₂ O, 10% CH ₃ CN, 0.25 M ammonium bicarbonate

B: 70% CH ₃ CN, 30% H ₂ O

Linear gradient: 75% A, 25% B to 45% A, 55% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 04_A4_1

UPLC (Method 04_A4_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 90% H ₂ O, 10% CH ₃ CN, 0.25 M ammonium bicarbonate

B: 70% CH ₃ CN, 30% H ₂ O

Linear gradient: 65% A, 35% B to 25% A, 65% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 08_B2_1

UPLC (Method 08_B2_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H ₂ O, 0.05% TFA

B: 99.95% CH ₃ CN, 0.05% TFA

Linear gradient: 95% A, 5% B to 40% A, 60% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 08_B4_1

UPLC (Method 08_B4_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H ₂ O, 0.05% TFA

B: 99.95% CH ₃ CN, 0.05% TFA

Linear gradient: 95% A, 5% B to 95% A, 5% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 05_B10_1

UPLC (Method 05_B10_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.).

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na ₂ SO ₄ , 0.04 MH ₃ PO ₄ , 10% CH ₃ CN (pH 3.5)

B: 70% CH ₃ CN, 30% H ₂ O

Linear gradient: 40% A, 60% B to 20% A, 80% B were used over 8 minutes at a flow rate of 0.40 ml/min.

Method 01_A4_2

UPLC (Method 01_A4_2): RP-analysis was performed using a Waters 600S system equipped with a Waters 996 diode array detector. UV detection at 214 nm and 254 nm was collected using a Symmetry300 C18, 5 um, 3.9 mm×150 mm column (42° C.). The HPLC system was connected to three eluent reservoirs containing: A: 100% H ₂ O, B: 100% CH ₃ CN, C: 1% trifluoroacetic acid in _{H 2 O.} Linear gradient: 90% A, 5% B, 5% C to 0% A, 95% B, 5% C were used over 15 minutes at a flow rate of 1.0 ml/min.

Method 09_B2_1

UPLC (Method 09_B2_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.). The UPLC system was connected to two eluent reservoirs containing: A: 99.95% H ₂ O, 0.05% TFA; B: 99.95% CH ₃ CN, 0.05% TFA. Linear gradient: 95% A, 5% B to 40% A, 60% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 09_B4_1

UPLC (Method 09_B4_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.). The UPLC system was connected to two eluent reservoirs containing: A: 99.95% H ₂ O, 0.05% TFA; B: 99.95% CH ₃ CN, 0.05% TFA. Linear gradient: 95% A, 5% B to 5% A, 95% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 05_B8_1

UPLC (Method 05_B8_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.). The UPLC system was connected to two eluent reservoirs containing: A: 0.2 M Na ₂ SO ₄ , 0.04 MH ₃ PO ₄ , 10% CH ₃ CN (pH 3.5); B: 70% CH ₃ CN, 30% H ₂ O. Linear gradient: 50% A, 50% B to 20% A, 80% B were used over 8 minutes at a flow rate of 0.40 ml/min.

Method 10_B14_1

UPLC (Method 10_B14_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH ShieldRP18, 1.7 um, 2.1 mm×150 mm column (50° C.). The UPLC system was connected to two eluent reservoirs containing: A: 99.95% H ₂ O, 0.05% TFA; B: 99.95% CH ₃ CN, 0.05% TFA. Linear gradient: 70% A, 30% B to 40% A, 60% B were used over 12 minutes at a flow rate of 0.40 ml/min.

Method 04_A6_1

UPLC (Method 04_A6_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.). The UPLC system was connected to two eluent reservoirs containing: A: 10 mM TRIS, 15 mM ammonium sulfate, 80% H ₂ O, 20%, pH 7.3; B: 80% CH ₃ CN, 20% H ₂ O. Linear gradient: 95% A, 5% B to 10% A, 90% B were used over 16 minutes at a flow rate of 0.35 ml/min.

Method 01_B4_1

HPLC (Method 01_B4_1): RP-analysis was performed using a Waters 600S system equipped with a Waters 996 diode array detector. UV detection was collected using a Waters 3 mm × 150 mm 3.5 um C-18 Symmetry column. The column was heated to 42° C. and eluted with a linear gradient of 5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in water over 15 minutes at a flow rate of 1 ml/min. .

Method 04_A7_1

UPLC (Method 04_A7_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.). The UPLC system was connected to two eluent reservoirs containing: A: 10 mM TRIS, 15 mM ammonium sulfate, 80% H ₂ O, 20%, pH 7.3; B: 80% CH ₃ CN, 20% H ₂ O. Linear gradient: 95% A, 5% B to 40% A, 60% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 05_B9_1

UPLC (Method 05_B9_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH130, C18, 130Å, 1.7 um, 2.1 mm×150 mm column (40° C.). The UPLC system was connected to two eluent reservoirs containing: A: 0.2 M Na ₂ SO ₄ , 0.04 MH ₃ PO ₄ , 10% CH ₃ CN (pH 3.5); B: 70% CH ₃ CN, 30% H ₂ O. Linear gradient: 70% A, 30% B to 20% A, 80% B were used over 8 minutes at a flow rate of 0.40 ml/min.

Method 10_B12_1

UPLC (Method 10_B12_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH ShieldRP18, 1.7 um, 2.1 mm×150 mm column (50° C.). The UPLC system was connected to two eluent reservoirs containing: A: 99.95% H ₂ O, 0.05% TFA; B: 99.95% CH ₃ CN, 0.05% TFA. Linear gradient: 50% A, 50% B to 0% A, 100% B were used over 16 minutes at a flow rate of 0.40 ml/min.

Method 04_A9_1

UPLC (Method 04_A9_1): RP-analysis was performed using a Waters UPLC system equipped with a dual band detector. UV detection at 214 nm and 254 nm was collected using an ACQUITY UPLC BEH Shield RP18, C18, 1.7 um, 2.1 mm×150 mm column (60° C.). The UPLC system was connected to two eluent reservoirs containing: A: 200 mM Na ₂ SO ₄ + 20 mM Na ₂ HPO ₄ + 20 mM NaH ₂ PO _{4 in 90% H 2 O / 10% CH 3} CN, pH 7.2; B: 70% CH ₃ CN, 30% H ₂ O. The following step gradient was used: 90% A, 10% B to 80% A, 20% B, 17 min over 3 min at a flow rate of 0.40 ml/min. Over 80% A, 20% B to 50% A, 50% B.

MALDI -MS method

Molecular weight was measured using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and recorded on Microflex or Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acid was used.

NMR method

Proton NMR spectra were recorded using Brucker Avance DPX 300 (300 MHz) with tetramethylsilane as an internal standard. The chemical shift (δ) is given in ppm and the partition pattern is referred to as: s, singlet; d, doublet; dd, double doublet; dt, double triplet; t, triplet; tt, triple triplet; q, quartet; quint, quintet; sext, sixt; m, multiplet; And br = wide.

B. Synthesis of intermediates

One. Fatty Synthesis of single ester

Overnight reflux of C12, C14, C16 and C18 diacids in toluene with Boc-anhydride, DMAP, and t- butanol predominately gives the t- butyl single ester. After the reaction, a mixture of monoacid, diacid and diester is obtained. Purification is carried out by washing, short plug silica filtration and crystallization.

B. Synthesis of the compounds of the present invention

Example One

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε37 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Arg ²⁶ ,Lys ²⁷ ,Arg ³⁴ ,Lys ³⁷ ]-GLP-1-(7-37)-peptide

Chem. 50:

Manufacturing method: SPPS method B

UPLC (Method 09_B2_1): Rt = 12.4min

UPLC (Method: 04_A3_1): Rt = 8.3min

LCMS4: Rt = 2.0 min, m/z = 1659 (m/3), 1244 (m/4), 996 (m/5)

Example 2

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Arg ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu-Gly

Chem. 51:

Manufacturing method: SPPS method B

UPLC (Method 09_B2_1): Rt = 13.1min

UPLC (Method 04_A7_1): Rt = 6.3min

LCMS4: Rt = 2.1 min, m/z = 1707 (m/3), 1280 (m/4), 1025 (m/5)

Example 3

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Arg ²⁶ ,Lys ²⁷ ,Arg ³⁴ ,Lys ³⁶ ],des-Gly37-GLP-1-(7-36)-peptide

Chem. 52:

Manufacturing method: SPPS method B

UPLC (Method 09_B2_1): Rt = 13.3 min

UPLC (Method 05_B5_1): Rt = 6.5min

LCMS4: Rt = 2.3 min, m/z = 1607 (m/3), 1205 (m/4), 964 (m/5)

Example 4

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε37 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Arg ³⁴ ,Lys ³⁷ ]-GLP-1-(7-37)-peptide

Chem. 53:

Manufacturing method: SPPS method B

UPLC (Method 09_B2_1): Rt = 13.0min

UPLC (Method 04_A7_1): Rt = 6.9 min

LCMS4: Rt = 2.0 min, m/z = 1668 (m/3), 1251 (m/4), 1001 (m/5)

Example 5

N ^ε20 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-] 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Lys ²⁰ ,Glu ²² ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Gly ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 54:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 9.02 min

UPLC (Method 04_A6_1): Rt = 4.61 min

LCMS4: Rt = 2.17 min, m/z = 1540 (m/3), 1155 (m/4)

Example 6

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Arg ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu

Chem. 55:

Manufacturing method: SPPS method B

UPLC (Method 09_B2_1): Rt = 13.0min

UPLC (Method 05_B5_1): Rt = 5.6min

LCMS4: Rt = 2.2 min, m/z = 1664 (m/3), 1248 (m/4), 999 (m/5)

Example 7

N ^ε22 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Lys ²² ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ , His ³¹ , Arg ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 56:

Manufacturing method: SPPS method B

LCMS2: Rt = 4.00 min, m/z = 1599 (m/3), 1199 (m/4)

UPLC (Method 08_B4_1): Rt = 7.83 min

UPLC (Method 05_B9_1): Rt = 7.45 min

Example 8

N ^ε22 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Lys ²² ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ , Arg ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 57:

Manufacturing method: SPPS method B

UPLC (Method 10_B12_1): Rt = 8.92 min

LCMS4: Rt = 2.58 min, m/z = 1525 (m/3), 1144 (m/4), 915 (m/5)

Example 9

N ^ε22 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib8,Lys22,Val25,Arg26,Lys27,Arg34]-GLP- 1-(7-35)-peptide

Chem. 58:

Manufacturing Method: SPPS Method E

The theoretical molecular mass of 4628 Da was confirmed by MALDI-MS.

UPLC (Method 09_B4_1): Rt = 9.29 min

UPLC (Method 04_A6_1): Rt = 6.49 min

Example 10

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[His ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Arg ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu

Chem. 59:

Manufacturing method: SPPS method B

UPLC (Method 08_B2_1): Rt = 12.9min

UPLC (Method 05_B5_1): Rt = 5.5min

LCMS4: Rt = 2.2 min, m/z = 1657 (m/3), 1243 (m/4), 995 (m/5)

Example 11

N ^ε22 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Lys ²² ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ , Gln ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 60:

Manufacturing method: SPPS method B

UPLC (Method 08_B2_1): Rt = 13.5min

LCMS4: Rt = 2.2 min, m/z = 1621 (m/3), 1216 (m/4), 973 (m/5)

Example 12

N ^α (N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino ]Butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[( 4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Arg ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl)-Gln

Chem. 61:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 8.9min

UPLC (Method 05_B7_1): Rt = 8.8 min

LCMS4: Rt = 2.2 min, m/z = 1673 (m/3), 1255 (m/4), 1004 (m/5)

Example 13

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Gln ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu

Chem. 62:

Manufacturing method: SPPS method B

UPLC (Method 05_B9_1): Rt = 7.9 min

UPLC (Method 05_B7_1): Rt = 8.8 min

LCMS4: Rt = 2.3 min, m/z = 1663 (m/3), 1248 (m/4), 999 (m/5)

Example 14

N ^ε22 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl ]Amino]ethoxy]ethoxy]acetyl]amino]butanoyl], N ^ε27 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2] -(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Lys ²² ,Arg ²⁶ ,Lys ²⁷ ,His ³¹ ,Gly ³⁴ ] -GLP-1-(7-34)-peptide

Chem. 63:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.5min

UPLC (Method 05_B7_1): Rt = 8.8 min

LCMS4: Rt = 2.1 min, m/z = 1462 (m/3), 1097 (m/4), 878 (m/5)

Example 15

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε37 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,His ³¹ , Gln ³⁴ , Lys ³⁷ ]-GLP-1-(7-37)-peptide

Chem. 64:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.3min

UPLC (Method 05_B9_1): Rt = 7.1 min

LCMS4: Rt = 2.1 min, m/z = 1589 (m/3), 1192 (m/4), 954 (m/5)

Example 16

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε37 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,His ³¹ ,Gln ³⁴ , Lys ³⁷ ]-GLP-1-(7-37)-peptide

Chem. 65:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.1 min

LCMS4: Rt = 2.1 min, m/z = 1585 (m/3), 1189 (m/4), 951 (m/5)

Example 17

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε37 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Glu ²³ ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ , His ³¹ , Gln ³⁴ , Lys ³⁷ ]-GLP-1-(7-37)-peptide

Chem. 66:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.0min

UPLC (Method 05_B9_1): Rt = 6.6 min

LCMS4: Rt = 2.1 min, m/z = 1609 (m/3), 1206 (m/4), 966 (m/5)

Example 18

N ^ε12 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Lys ¹² ,Glu ²² ,Arg ²⁶ ,Lys ²⁷ , His ³¹ ,Gln ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 67:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 7.66min

UPLC (Method 04_A6_1): Rt = 4.09 min

LCMS4: Rt=1.83 min, m/z = 1181 (m/4), 945 (m/5)

Example 19

N ^ε22 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Lys ²² ,Arg ²⁶ ,Lys ²⁷ ,His ³¹ , Gly ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 68:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 8.37 min

UPLC (Method 04_A6_1): Rt = 4.41 min

LCMS4: Rt = 2.00 min, m/z = 1466 (m/3), 1100 (m/4)

Example 20

N ^ε22 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl ]Amino]ethoxy]ethoxy]acetyl]amino]butanoyl], N ^ε27 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2] -(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Lys ²² ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ , Gln ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 69:

Manufacturing Method: SPPS Method E

UPLC (Method 09_B4_1): Rt = 9.00 min

UPLC (Method 04_A6_1): Rt = 6.50 min

LCMS4: Rt = 2.23 min, m/z = 1215 (m/4), 972 (m/5)

Example 21

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,His ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Arg ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu

Chem. 70:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 8.4min

UPLC (Method 04_A6_1): Rt = 9.3 min

LCMS4: Rt = 2.2 min, m/z = 1681 (m/3), 1261 (m/4), 1009 (m/5)

Example 22

N ^ε24 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl ]Amino]ethoxy]ethoxy]acetyl]amino]butanoyl], N ^ε27 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2] -(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Glu ²² ,Lys ²⁴ ,Arg ²⁶ ,Lys ²⁷ ,His ³¹ , Gly ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 71:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 8.17 min

UPLC (Method 04_A6_1): Rt = 4.65 min

LCMS4: Rt = 1.98 min, m/z = 1481 (m/3), 1111 (m/4)

Example 23

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[10-( 4-carboxyphenoxy)decanoylamino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2] -[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]buta Noyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Gln ³⁴ ,Lys ³⁶ ]-GLP-1-( 7-37)-peptide

Chem. 72:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.82 min

UPLC (Method 05_B5_1): Rt = 6.10 min

LCMS4: Rt = 2.37 min, m/z = 1687 (m/3), 1266 (m/4), 1013 (m/5)

Example 24

N ^ε24 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Lys ²⁴ ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ , His ³¹ , Arg ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 73:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 7.52 min

UPLC (Method 04_A9_1): Rt = 10.35 min

LCMS4: Rt = 1.92 min, m/z = 1613 (m/3), 1210 (m/4), 968 (m/5), 807 (m/6)

Example 25

N ^ε24 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-] 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Lys ²⁴ ,Arg ²⁶ ,Lys ²⁷ ,His ³¹ ,Gly ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 74:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.01 min

UPLC (Method 04_A9_1): Rt = 8.00 min

LCMS4: Rt = 2.08 min, m/z = 1513 (m/3), 1135 (m/4), 908 (m/5)

Example 26

N ^ε27 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4R)]-4-carboxy-4-[10-( 4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl], N ^ε36 -[(4S)-4-carboxy -4-[[2-[2-[2-[[2-[2-[2-[[(4R)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]buta Noyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Gln ³⁴ ,Lys ³⁶ ]-GLP-1-( 7-37)-peptide

Chem. 75:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 8.96 min UPLC (Method 05_B5_1): Rt = 6.54 min

UPLC (Method 04_A6_1): Rt = 5.69 min

LCMS4: m/z: Rt = 3.07 min, m/z = 1687 (m/3), 1266 (m/4), 1013 (m/5)

Example 27

N ^ε24 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Lys ²⁴ ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ , Gly ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 76:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 9.25 min

UPLC (Method 04_A6_1): Rt = 6.01 min

LCMS4: Rt = 3.31 min, m/z = 1506 (m/3), 1130 (m/4), 4520 (m/5)

Example 28

N ^ε24 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-] 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² ,Lys ²⁴ ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ ,Arg ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 77:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 8.19 min

UPLC (Method 04_A6_1): Rt = 5.24 min

LCMS4: Rt = 3.22 min, m/z = 1663 (m/3), 1247 (m/4), 998 (m/5), 832 (m/6)

Example 29

N ^ε24 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy ]Ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4] -(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Glu ²² ,Lys ²⁴ ,Val ²⁵ ,Arg ²⁶ , Lys ²⁷ , His ³¹ , Gln ³⁴ ]-GLP-1-(7-37)-peptide

Chem. 78:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 7.79 min

UPLC (Method 04_A6_1): Rt = 4.87 min

Example 30

N ^ε24 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl ]Amino]ethoxy]ethoxy]acetyl]amino]butanoyl], N ^ε27 -[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2] -(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Glu ²² ,Lys ²⁴ ,Val ²⁵ ,Arg ²⁶ ,Lys ²⁷ , Gly ³⁴ ]-GLP-1-(7-34)-peptide

Chem. 79:

Manufacturing method: SPPS method B

UPLC (Method 08_B4_1): Rt = 9.33 min

UPLC (Method 04_A6_1): Rt = 6.13 min

LCMS4: Rt = 2.98 min, m/z = 1506 (m/3), 1130 (m/4), 904 (m/5)

Example 31

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl ]Amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)]- 4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib ⁸ ,Glu ²² ,Arg ²⁶ ,Lys ²⁷ ,Glu ³⁰ ,Arg ³⁴ ,Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu-Gly

Chem. 80:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1):_Rt = 8.58min UPLC (Method 10_B29_1): Rt = 10.6min

UPLC (Method 04_A6_1): Rt = 4.43 min

LCMS4: Rt = 3.72 min; m/3: 1712; m/4: 1284; m/5: 1028

Example 32

N ^ε27 -[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]buta Noyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl], N ^ε36 -[2-[2-[2-[[2-[2-[2-[[(4S)] -4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu ²² , Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Arg ³⁴ , Lys ³⁶ ]-GLP-1-(7-37)-peptidyl-Glu-Gly

Chem. 81:

Manufacturing method: SPPS method B

UPLC (Method 09_B4_1): Rt = 9.19 min

UPLC (Method 10_B29_1): Rt = 13.73 min

UPLC (Method 04_A6_1): Rt = 5.40 min

LCMS4: Rt = 2.44 min; m/3: 1726; m/4: 1294; m/5:1036

Pharmacological method

Example 33: In vitro efficacy

The purpose of this example is to test the activity or efficacy of GLP-1 derivatives in vitro.

The efficacy of the GLP-1 derivatives of Examples 1-32 is determined as described below, i.e. as stimulation of cyclic AMP (cAMP) formation in a medium containing a membrane expressing the human GLP-1 receptor.

principle

Plasma membranes purified from BHK467-12A (tk-ts13), a stable transfected cell line expressing human GLP-1 receptors, were stimulated with the corresponding GLP-1 analogs or derivatives, and the efficacy of cAMP production was determined by AlphaScreenTM from Perkin Elmer Life Sciences. It was measured using the cAMP Assay Kit. The basic principle of the AlphaScreen Assay is the competition between endogenous cAMP and exogenously added biotin-cAMP. Capture of cAMP is achieved by using specific antibodies conjugated to receptor beads.

Cell culture and membrane preparation

Stable transfected cell lines and high expressing clones were selected for testing. Cells were grown in DMEM, 5% FCS, 1% Pen/Strep (penicillin/streptomycin) and 0.5 mg/ml of selectable marker G418 in 5% CO _2.

Cells of about 80% confluence were washed twice with PBS (aqueous solution of tetrasodium salt of ethylenediaminetetraacetic acid), harvested, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. All further steps were performed on ice. The cell pellet was homogenized by Ultrathurax for 20-30 seconds in 10 ml of buffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged for 15 minutes at 20,000 rpm, and the pellet was subjected to 10 ml of buffer 2 (20 mM Na-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenized for 20-30 seconds and centrifuged for 15 minutes at 20,000 rpm. Suspension, homogenization and centrifugation were repeated once in buffer 2 and the membrane was resuspended in buffer 2. The protein concentration was measured and stored at -80°C until the membrane was used.

The assay was performed in a flat bottom 96-well plate (Costar cat. no:3693). The final volume per well was 50 μl.

Solutions and reagents

AlphaScreen cAMP Assay from Perkin Elmer Life Sciences, containing Anti-cAMP receptor beads (10 U/μl), streptavidin donor beads (10 U/μl) and biotinylated-cAMP (133 U/μl) Kit (cat.No: 6760625M).

AlphaScreen buffer, pH=7.4: 50 mM TRIS-HCl (Sigma, cat.no: T3253); 5 mM HEPES (Sigma, cat.no: H3375); 10 mM MgCl ₂ , 6H ₂ O (Merck, cat.no: 5833); 150 mM NaCl (Sigma, cat.no: S9625); 0.01% Tween (Merck, cat.no: 822184). The following were added to AlphaScreen buffer prior to use (expressed as final concentration): BSA (Sigma, cat. no. A7906): 0.1%; IBMX (Sigma, cat. no. I5879): 0.5 mM; ATP (Sigma, cat. no. A7699): 1 mM; GTP (Sigma, cat. no. G8877): 1 uM.

cAMP standard (dilution factor in the assay = 5): cAMP solution: 5 μl of 5 mM cAMP-stock solution + 495 μl AlphaScreen buffer.

The GLP-1 analogues or derivatives to be tested are, for example: 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^-13 and A suitable dilution series of 8 concentrations of 10 ^-14 M of the GLP-1 compound as well as AlphaScreen buffer of the cAMP standard were prepared, for example, with a series from cAMP of ^{10 -6} to 3×10 ^-11.

Membrane/receptor Bead

Membranes were prepared from hGLP-1/BHK 467-12A cells with a concentration of 6 μg/well corresponding to 0.6 mg/ml (the amount of membrane used per well may vary).

"No membrane": receptor beads in AlphaScreen buffer (final 15 μg/ml)

"6 μg/well of membrane": membrane + receptor beads in AlphaScreen buffer (final 15 μg/ml)

An aliquot (10 μl) of “no membrane” was added to the cAMP standard (per well of the second well) and positive and negative controls.

An aliquot (10 μl) of “6 μg/well membrane” was added to GLP-1 and analogs (per well of the second or third well).

Positive control: 10 μl "no membrane" + 10 μl AlphaScreen buffer

Negative control: 10 μl "no membrane" + 10 μl cAMP stock solution (50 μM)

Because the beads are sensitive to direct sunlight, some handling was done in the dark (as dark as possible), or in green light. All dilutions were done on ice.

process

1. Make AlphaScreen Buffer.

2. Dissolving and diluting the GLP-1/analog/cAMP standard in AlphaScreen buffer.

3. Make a donor bead solution by mixing streptavidin donor beads (2 units/well) and biotinylated cAMP (1.2 units/well) and incubate for 20-30 minutes in a dark room at room temperature.

4. Add cAMP/GLP-1/analog to plate: 10 μl per well.

5. Make a membrane/receptor bead solution and add it to the plate: 10 μl per well.

6. Add donor beads: 30 μl per well.

7. Wrap the plate in aluminum foil and incubate (very slowly) on a shaker for 3 hours at RT.

8. Counting on AlphaScreen-Pre-incubate each plate on AlphaScreen for 3 minutes prior to counting.

result

EC ₅₀ [pM] values were calculated using Graph-Pad Prism software (version 5) and are shown in Table 1 below. In vitro efficacy of all derivatives was confirmed.

In vitro efficacy Example Number of compounds EC ₅₀ /pM One 26 2 43 3 62 4 143 5 468 6 96 7 9 8 159 9 242 10 214 11 81 12 41 13 79 14 42 15 5 16 21 17 17 18 3025 19 52 20 67 21 52 22 1178 23 140 24 70 25 380 26 200 27 835 28 68 29 40 30 3000 31 37 32 76

The average in vitro efficacy (EC ₅₀ mean) for the tested compounds was 340 pM. Most of the derivatives had good in vitro potency corresponding to _{an EC 50 below 1200 pM.}

For comparison, see Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. Compound 13 of Table 1, 1664-669 (a bis -C12- discrete Oh the GLP-1 (7-37 misfire) in K ^26,34) had an in vitro efficacy which corresponds to 1200 pM of the EC _50.

Example 34: GLP-1 receptor binding

The purpose of this experiment is to investigate the binding of GLP-1 derivatives to the GLP-1 receptor and how binding is potentially affected by the presence of albumin. This is done in an in vitro experiment as described below.

The binding affinity of the GLP-1 derivatives of Examples 1-32 to the human GLP-1 receptor was measured by their ability to displace ^{125 I-GLP-1 from the receptor.} To test the binding of the derivative to albumin, the assay was performed with a high concentration of albumin (2.0% addition), as well as a low concentration of albumin (0.001%-corresponding to its residual amount in the tracer). The shift in binding affinity, which is IC ₅₀ , is an indication that the peptide in question binds to albumin, thereby predicting the potential extended to the pharmacokinetic profile of the peptide in question in an animal model.

Condition

Species (in vitro): Hamster

Biological termination point: receptor binding

Method Method: SPA

Receptor: GLP-1 receptor

Cell line: BHK tk-ts13

Cell culture and membrane purification

Stable transfected cell lines and high expressing clones were selected for testing. Cells were grown in DMEM, 10% FCS, 1% Pen/Strep (penicillin/streptomycin) and 1.0 mg/ml of selectable marker G418 in 5% CO _2.

Cells (about 80% confluence) were washed twice in PBS and harvested with Versene (aqueous solution of tetrasodium salt of ethylenediaminetetraacetic acid), and they were separated by centrifugation at 1000 rpm for 5 minutes. Cells/cell pellets should be stored on ice to the extent possible for subsequent steps. Cell pellets were homogenized with Ultrathurrax for 20-30 seconds in an appropriate amount of buffer 1 (depending on the amount of cells, but for example 10 ml). The homogenate was centrifuged at 20000 rpm for 15 minutes. The pellet was resuspended (homogenized) in 10 ml buffer 2 and centrifuged again. This step was repeated one more time. The resulting pellet was resuspended in buffer 2, and the protein concentration was measured. The membrane was removed and stored at 80°C.

Buffer 1: 20 mM Na-HEPES + 10 mM EDTA, pH 7.4

Buffer 2: 20 mM Na-HEPES + 0.1 mM EDTA, pH 7.4

Binding Assay:

SPA:

Test compound, membrane, SPA-particles and [ ¹²⁵ I]-GLP-1(7-36)NH ₂ were diluted in assay buffer. 50 ul (microliter) HSA ("high albumin" experiment containing 2% HSA), or buffer ("low albumin" experiment containing 0.001% HSA) was added to Optiplate, and 25 ul of test compound was added. . 5-10 ug membrane protein/sample (50 ul) corresponding to 0.1-0.2 mg protein/ml (preferably optimized for each membrane preparation) were added. SPA-particles (Wheatgerm agglutinin SPA beads, Perkin Elmer, #RPNQ0001) were added in an amount of 0.5 mg/well (50 ul). Culture was started with [ ¹²⁵ I]-GLP-1]-(7-36)NH ₂ (final concentration 0.06 nM, 25 ul corresponding to 49.880 DPM). The plate was sealed with PlateSealer and incubated at 30° C. for 120 minutes while shaking. The plate was centrifuged (1500 rpm, 10 minutes) and counted in a Topcounter.

Assay buffer :

50 mM HEPES

5 mM EGTA

5 mM MgCl ₂

0.005% Tween 20

pH 7.4

HSA is SIGMA A1653

Calculation

The IC ₅₀ value is read from the curve as the concentration that displaces 50% of ¹²⁵ _{I-GLP-1 from the receptor, and the ratio of [(IC 50} /nM) high HSA] / [(IC ₅₀ /nM) low HSA]. I decided.

In general, binding to the GLP-1 receptor at low albumin concentrations is as good as possible, and should correspond to _{low IC 50 values.}

_{The IC 50} value at high albumin concentration is a measure of the influence of albumin on the binding of the derivative to the GLP-1 receptor. As is known, the GLP-1 derivative also binds to albumin. This is generally a desirable effect of extending their lifespan in plasma. Thus, IC ₅₀ values at high albumin are generally more than _{IC 50} values at low albumin, corresponding to reduced binding to the GLP-1 receptor, caused by albumin binding that competes with binding to the GLP-1 receptor. Will be high.

Therefore, a high ratio (IC ₅₀ value (high albumin) / IC ₅₀ value (low albumin)) binds the derivative well to albumin (may have a long half-life), and also binds itself well to the GLP-1 receptor. (IC ₅₀ value (high albumin) is high, IC ₅₀ value (low albumin) is low) can be taken as an indication.

result

The following results were obtained, where "ratio" refers to [(IC ₅₀ /nM) high HSA] / [(IC ₅₀ /nM) low HSA]):

Receptor binding affinity Example Number of compounds IC ₅₀ /nM
(Low HSA) IC ₅₀ /nM
(High HSA) ratio
One 0.19 42 219 2 0.39 320 821 3 0.29 29 101 4 0.15 33 217 5 5.68 446 79 6 0.50 123 246 7 0.12 21 174 8 1.41 80 57 9 0.38 60 157 10 6.49 452 70 11 0.23 213 926 12 0.16 72 453 13 0.25 201 804 14 1.45 443 306 15 0.19 29 155 16 0.20 25 127 17 0.45 174 387 18 373 789 2,1 19 2.38 143 60 20 0.19 333 1752 21 1.57 256 163 22 9.31 812 87 23 0.85 40 47 24 2.74 50 18 25 10.9 39 3,5 26 0.38 54 143 27 10.3 >1000 97 28 0.20 29 144 29 3.95 363 92 30 7.44 >1000 134 31 0.35 220 629 32 0.18 369 2050

The average proportion was very good (about 300). Most of the derivatives had a ratio above 50.

Moreover _{, with respect to IC 50} (low albumin), the average IC ₅₀ of the tested compounds was 14 nM, and most of the derivatives were below 15.0 nM.

Finally, _{with respect to the IC 50} (high albumin), most of the derivatives had an IC ₅₀ (high albumin) below 900 nM.

For comparison, see Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. Compound 13 of Table 1, 1664-669 (a bis -C12- discrete acylated GLP-1 (7-37) in the K ^26,34) is the ratio of 51.3, in 17.7 nM IC ₅₀ (low albumin), and It had an IC ₅₀ (high albumin) of 908 nM.

Example 35: Estimation of oral bioavailability- On the rat Intestinal injection ( Caprate )

The purpose of this experiment is to estimate the oral bioavailability of GLP-1 derivatives.

To this end, exposure to plasma after direct injection of the GLP-1 derivatives of Examples 2-17 and 19-22 into the intestinal lumen was studied in vivo in rats, as described below.

The GLP-1 derivative was tested at a concentration of 1000 uM in 55 mg/ml sodium caprate solution.

Male Sprague Dawley rats weighing about 240 g upon arrival were obtained from Taconic (Denmark) and 4 rats per group were assigned by simple randomization for different treatments. Rats were fasted for about 18 hours prior to the experiment and under normal anesthesia (hyphnom/domicum).

GLP-1 derivatives were administered in the jejunum to the proximal portion (10 cm distal to the duodenum) or mid-intestine (50 cm proximal to the cecum). A 10 cm long PE50-catheter was inserted into the jejunum, at least 1.5 cm was placed in the jejunum, and the intestinal and catheter circumference was fixed by 3/0 sutures end-to-end ligation before dosing to prevent leakage or catheter displacement. The catheter was placed without a syringe and needle, and 2 ml saline was administered to the abdomen, and the incision was closed with a wound clip.

100 μl of each GLP-1 derivative was injected into the jejunum lumen through a catheter with a 1 ml syringe. Then, 200 μl of air was pushed into the jejunum lumen with another syringe to “flush” the catheter. This syringe was left connected to the catheter to prevent reflux into the catheter.

Blood samples (200 ul) were collected from the tail vein into the EDTA tube at the desired intervals (typically times 0, 10, 30, 60, 120 and 240 minutes) and centrifuged for 5 minutes at 10000 G at 4° C. within 20 minutes. Plasma (75 ul) is generally used for measurement of insulin in Poulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12, p. As described as 240-247, the plasma concentrations of each GLP-1 derivative were immediately frozen and held at -20°C until analyzed by LOCI (Lumescent Oxygen Channeling Immunoassay), separated into Micronic tubes. The donor beads were coated with streptavidin, while the receptor beads were conjugated with a monoclonal antibody that recognized the mid-/C-terminal epitope of the peptide. Another monoclonal antibody specific for the N-terminus was biotinylated. The three reactions were combined with the analyte and a two-site immuno-complex was formed. Illumination of the complex released a singlet oxygen atom from the donor beads channeled to the receptor beads and triggered by chemiluminescence measured with an Envision plate reader. The amount of light was proportional to the concentration of the compound.

After blood sampling, rats were sacrificed under anesthesia and the abdomen was opened to confirm correct catheter placement.

The mean (n=4) plasma concentration (pmol/l) was measured as a function of time. The ratio of the plasma concentration (pmol/l) divided by the concentration of the dosing solution (μMol/l) was calculated for each treatment, and the results for t = 30 minutes (30 minutes after injection of the compound into the plant) were calculated as a surrogate of intestinal bioavailability. Assessed as a scale (dose-corrected exposure at 30 minutes). Dose-corrected exposure has been shown to correlate significantly with actual bioavailability.

The following results were obtained when the dose-corrected exposure at 30 minutes referred to (plasma concentration (pM) 30 minutes after injection of the compound to the plant), divided (concentration of the compound in the dosing solution (μM)):

Dose-corrected exposure at 30 minutes Example Number of compounds In 30 minutes
Dose-corrected exposure 2 98 3 67 4 39 5 124 6 93 7 99 8 86 9 65 10 187 11 66 12 68 13 126 14 121 15 98 16 115 17 168 19 61 20 123 21 140 22 275

All derivatives had a dose-corrected exposure of at least 38 at 30 minutes.

For comparison, see Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. Compound 13 (a bis -C12- discrete acylated GLP-1 (7-37) in the K ^26,34) of Table 1 is more than 38 1664-669 capacity at 30 minutes had a corrected exposure.

Example 36: blood Glucose And effect on body weight

The purpose of this study was to confirm the effect of GLP-1 derivatives on blood glucose (BG) and body weight (BW) in the setting of diabetic patients.

The GLP-1 derivative is tested in a dose-response study of an obese, diabetic mouse model (db/db mice) as described below.

7-9 db/db mice (Taconic, Denmark) fed with diet NIH31 (NIH 31M Rodent Diet, available commercially from Taconic Farms, Inc., US, see www.taconic.com) from birth. Registered for study at the age of the week. Mice have free access to standard food (eg Altromin 1324, Brogaarden, Gentofte, Denmark) and tap water and are maintained at 24°C. After 1-2 weeks of acclimatization, basal blood glucose is assessed twice in two consecutive days (i.e. at 9 am). Mice with the lowest blood glucose value are excluded from the experiment. Based on the mean blood glucose value, the remaining mice are selected for further experiments and assigned to 7 groups (n=6) matching blood glucose levels. Mice are used for a period of 48 hours and for up to 4 experiments. Mice are euthanized after the last experiment.

The 7 groups receive the following treatment:

1: vehicle, subcutaneous

2: GLP-1 derivative, 0.3 nmol/kg, subcutaneous

3: GLP-1 derivative, 1.0 nmol/kg, subcutaneous

4: GLP-1 derivative, 3.0 nmol/kg, subcutaneous

5: GLP-1 derivative, 10 nmol/kg, subcutaneous

6: GLP-1 derivative, 30 nmol/kg, subcutaneous

7: GLP-1 derivative, 100 nmol/kg, subcutaneous

Vehicle: 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4.

The GLP-1 derivative is dissolved in the vehicle at concentrations of 0.05, 0.17, 0.5, 1.7, 5.0 and 17.0 nmol/ml. Animals are administered subcutaneously at a dose-volume of 6 ml/kg (ie 300 μl per 50 g mouse).

On the dosing day, mice are weighed and blood glucose assessed at time -½ hours (8.30 am). The GLP-1 derivative is dosed at about 9 am (time 0). On the dosing day, blood glucose is assessed at times 1, 2, 4 and 8 hours (10 am, 11 am, 1 pm and 5 pm). After 8 hours of blood sampling, the mice were weighed.

On the next day, blood glucose is assessed at hours 24 and 48 post dosing (ie, 9 am on days 2 and 3). On each day, mice are weighed after blood glucose sampling.

Each mouse is weighed on a digital scale.

Samples for measurement of blood glucose are obtained from capillaries at the tail end of a conscious mouse. 10 μl of blood is collected by heparin capillary and transferred to 500 μl glucose buffer (EKF system solution, Eppendorf, Germany). Glucose concentration is measured using the glucose oxidase method (glucose analyzer Biosen 5040, EKF Diagnostic, GmbH, Barleben, Germany). Samples are kept at room temperature for up to 1 hour until analysis. If the analysis should be postponed, the sample is held at 4° C. for up to 24 hours.

ED ₅₀ is the dose that produces the half-maximal effect in nmol/kg. This value is calculated based on the ability of the derivative to lower body weight as well as the ability to lower blood glucose, as described below.

_{ED 50} for body weight is calculated as the dose that produces a half-maximal effect at 8 hours delta BW after subcutaneous administration of the derivative. For example, if the maximum loss of body weight after 8 hours is 2.0 g, then the ED ₅₀ body weight is the dose that results in 1.0 g of body weight loss after 8 hours. This dose (ED ₅₀ body weight) can be read from the dose-response curve.

_{ED 50} for blood glucose is calculated as the dose that produces a half-maximal effect at 8 hours and/or 24 hours of AUC delta BG after subcutaneous administration of the analog.

The ED ₅₀ value can only be calculated if a suitable sigmoidal dose-response relationship exists with a clearly defined maximum response. Thus, if this is not the case, the derivative can be retested in a different range of doses to see that a sigmoidal dose-response relationship is obtained.

Example 37: In a mini pig Half-life

The purpose of this study was to measure the delay of their action time after intravenous administration to mini pigs. This is done in a pharmacokinetic (PK) study, which measures the final half-life of the derivative in question. Terminal half-life generally refers to the period of time it takes to halve a specific plasma concentration measured after the initial distribution.

Male Gottingen mini-pigs were obtained from Ellegaard Gottingen mini-pigs (Dalmose, Denmark), and those aged about 7-14 months and weighing about 16-35 kg were used in the study. Each of the mini pigs was housed and the SDS mini pig diet (Special Diets Services, Essex, UK) was fed limitedly once or twice. After at least 2 weeks of acclimatization, two permanent central venous catheters were implanted in each animal's vena cava head or tail. Animals were recovered one week post-surgery and then used in repeated pharmacokinetic studies with a suitable washout period between successive GLP-1 derivative dosing.

Animals were fasted for about 18 hours before dosing and 0-4 hours after dosing, but had free access to water for the entire period.

The GLP-1 derivative was dissolved in 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4 at a concentration of 20-60 nmol/ml. Intravenous injection of the compound (typically a volume equivalent to 1-2 nmol/kg, e.g. 0.033 ml/kg) is given through one catheter, and the blood is administered post-dose (preferably through another catheter) It was sampled at a fixed time point up to one day or less. Blood samples (eg 0.8 ml) were collected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 1942 G for 10 minutes. Plasma was pipetted into Micronic tubes on dry ice and the plasma concentration of each GLP-1 compound was maintained at -20°C until analyzed using ELISA or similar antibody based assays or LC-MS. Individual plasma concentration-time profiles were obtained from Phoenix WinNonLin ver. Analysis by non-compartmental model of 6.2. (Pharsight Inc., Mountain View, CA, USA), and the resulting terminal half-life (harmonized mean) was determined.

result

The derivative of Example 2 was tested and it had a half-life of 87 hours.

For comparison, see Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. (In the ah K ^26,34 as bis -C12- discrete acylated GLP-1 (7-37)) Compound No. 13 in Table 1 of the 1664-669 had a half-life of 5 hours.

Example 38: Effects on food intake

The purpose of this experiment was to investigate the effect of GLP-1 derivatives on the food intake of pigs. This was performed in a pharmacodynamic (PD) study in which food intake was measured 1 to 4 days after administration of a single dose of the GLP-1 derivative compared to the vehicle-treated control as described below.

Female Landrace Yorkshire Duroc (LYD) pigs of about 3 months of age and weighing about 30-35 kg were used (n=3-4 per group). Animals were housed in groups for about 1 week during acclimation to the animal facility. During the experiment, animals were placed in individual fences at least 2 days prior to dosing and throughout the entire experiment for measurement of individual food intake. Animals were fed freely with pig feed (Svinefoder Danish Top) at all times both during acclimatization and experimentation. Food intake was monitored by recording the weight of the feed online every 15 minutes. As the system, Mpigwin (Ellegaard Systems, Faaborg, Denmark) was used.

The GLP-1 derivative was added to a phosphate buffer (50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4) at a dose of 0.3, 1, 3, 10 or 30 nmol/kg, equivalent to 12, 40, 120, 400. Alternatively, it is dissolved at a concentration of 1200 nmol/ml. The phosphate buffer acts as a vehicle. Animals are dosed as a single subcutaneous dose of GLP-1 derivative or vehicle (dose volume 0.025 ml/kg) on the morning of Day 1, and food intake is measured for 1-4 days after dosing. On the last day of each study, 1-4 days after dosing, a blood sample for measurement of plasma exposure of the GLP-1 derivative is taken from the heart of an anesthetized animal. The animals are then euthanized with an overdose of pentobarbitone in the heart. Plasma content of GLP-1 derivatives is analyzed using ELISA or similar antibody based assays, or LC-MS.

Food intake is calculated as the mean ± SEM 24 hour food intake on each experimental day. Statistical comparison of 24-hour food intake versus GLP-1 derivative group in vehicle is performed using binary-ANOVA repeated measurements followed by Bonferroni post-test.

The compound of Example 10 was tested at a dose of 3 nmol/kg, and there was no effect on food intake at this dose. The compound of Example 2 was tested at a dose of 3 nmol/kg and a significant decrease in food intake was observed on all days of the experiment (23% on day 1, and 35% on day 2).

Example 39: In rats Pharmacokinetics

The purpose of this example is to investigate the in vivo half-life of rats.

In vivo pharmacokinetic studies in rats were performed with the GLP-1 derivatives of Examples 2, 10, 17-18, and 31, as described below. Semaglutide was included for comparison.

Male Sprague Dawley rats of the same age with a body weight of about 400 g were obtained from Taconic (Denmark), and about 4 rats per group in body weight were assigned to treatment by simple randomization, so that all animals in each group were of similar body weight.

The GLP-1 derivative (about 6 nmol/ml) was dissolved in 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4. Intravenous injection of the compound (1.0 ml/kg) was given through a catheter implanted in the right jugular vein. Blood was sampled from the sublingual vein for 5 days post-dose. Blood samples (200 μl) were collected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 10000 G for 5 minutes. Plasma samples were maintained at -20°C until the plasma concentration of each GLP-1 compound was analyzed.

Plasma concentrations of GLP-1 compounds are generally determined by Poulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12, p. As described for the measurement of insulin by 240-247, measurements were made using luminescent oxygen channeling immunoassay (LOCI). The donor beads were coated with streptavidin, while the receptor beads were conjugated with a monoclonal antibody that recognized the mid-/C-terminal epitope of the peptide. Another monoclonal antibody specific for the N-terminus was biotinylated. The three reactions were combined with the analyte and a two-site immuno-complex was formed. Illumination of the complex released a singlet oxygen atom from the donor beads channeled to the receptor beads and triggered by chemiluminescence measured with an Envision plate reader. The amount of light was proportional to the concentration of the compound.

Plasma concentration-time profiles were obtained from Phoenix WinNonLin ver. Analysis was performed using 6.2 (Pharsight Inc., Mountain View, CA, USA), and half-life (T _½ ) was calculated using individual plasma concentration-time profiles from each animal.

result

The half-life of semaglutide tested in the same setup (but n=8) was 11 hours.

In rats Half-life Example Number of compounds T½ / hour 2 26 10 23 17 10 18 10 31 19

The tested derivatives of the present invention had a half-life similar to or better than semaglutide.

Example 40: Estimation of oral bioavailability- On the rat Enteral injection and oral forced feeding (SNAC)

The purpose of this experiment is to estimate the oral bioavailability of GLP-1 derivatives in a rat model.

Briefly, sodium N of the GLP-1 derivative - [8- (2-hydroxybenzoyl) amino] caprylate (the previous version) Secretary scanning the liquid solution in (SNAC), or administered by oral feeding force (up) , The subsequent exposure to plasma of the GLP-1 derivative is measured.

A stock solution of 250 mg/ml SNAC is prepared by dissolving SNAC (12.5 g) in high pure laboratory water (MilliQ) (50.0 ml). The pH is adjusted to about 8.5 with 1 N NaOH (aqueous solution).

A solution of about 1000 uM (800-1200 uM) GLP-1 derivative in 250 mg/ml SNAC is prepared by dissolving the desired amount of each GLP-1 derivative in the SNAC stock solution. The concentration of the GLP-1 derivative is determined prior to administration by state-of-the-art methods such as CLND-HPLC (chemiluminescent nitrogen detection for HPLC).

Thirty-two male Sprague Dawley rats weighing about 240 g upon arrival were obtained from Taconic (Denmark) and assigned 8 rats per group by simple randomization for other treatments. All rats are fasted on the grid for about 18 hours prior to the experiment.

For intestinal injection , on the day of the experiment, rats are taken under general anesthesia (hyphnom/domicum) and anesthetized throughout the entire experiment. The GLP-1 derivatives of Examples 5-7 were administered to the proximal portion of the jejunum (10 cm end to the duodenum). A 10 cm long PE50-catheter is inserted into the jejunum, at least 1.5 cm is placed in the jejunum, and the intestinal and catheter circumference is fixed by 3/0 sutures end-to-end ligation prior to dosing to prevent leakage or catheter displacement. Moreover, the catheter has a 3/0 suture from end to end to prevent leakage or catheter displacement. The catheter is placed without a syringe and needle, 2 ml saline is administered to the abdomen, and the incision is closed with a wound clip.

100 μl of the SNAC solution of each GLP-1 derivative is injected into the jejunum lumen through a catheter with a 1 ml syringe. Then, 200 μl of air is pushed into the jejunum lumen with another syringe to “flush” the catheter. This syringe is left connected to the catheter to prevent reflux into the catheter.

Blood samples (200 ul) are collected from the tail vein into the EDTA tube at the desired intervals (typically times 0, 10, 30, 60, 120 and 180 minutes).

For forced oral feeding , animals are conscious throughout the entire experiment.

100 μl of the SNAC solution of the GLP-1 derivative is administered by oral forced feeding directly into the stomach.

Blood samples (200 ul) are collected from the sublingual plexus to the EDTA tube at desired intervals (typically times 0, 10, 30, 60, 120 and 180 minutes).

All obtained blood samples are kept on ice and centrifuged for 5 minutes at 10000 G at 4° C. within 20 minutes. Plasma (75 ul) is generally used for measurement of insulin in Poulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12, p. As described as 240-247, the plasma concentrations of each GLP-1 derivative were immediately frozen and maintained at -20°C until analyzed by LOCI (Lumescent Oxygen Channeling Immunoassay), separated into Micronic tubes. While the donor beads were coated with streptavidin, the receptor beads were conjugated with a monoclonal antibody that recognized the mid-/C-terminal epitope of the peptide. Another monoclonal antibody specific for the N-terminus is biotinylated. The three reactions are combined with the analyte and a two-site immuno-complex is formed. Illumination of the complex released a singlet oxygen atom from the donor beads channeled to the receptor beads and triggered by chemiluminescence measured with an Envision plate reader. The amount of light is proportional to the concentration of the compound.

After blood sampling, all rats are sacrificed under anesthesia and the abdomen of intestinal injection rats is opened to confirm correct catheter placement.

The mean (n=8) plasma concentration (pmol/l) is determined as a function of time. The AUC (pmol/l) versus time curve of plasma exposure from time 30-180 (min) is dose-corrected, ie divided by the amount (dose) (pmol) in the administered solution of the derivative. Thus, the dose-corrected AUC of plasma exposure from time 30-180 min (with units of minutes × pM / pmol = min/L) is a surrogate measure of bioavailability-ranking the derivative for its absorption in the rat model. Is used as a measure.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Accordingly, it is believed that the appended claims are intended to cover all such modifications and variations that fall within the true concept of the invention.

SEQUENCE LISTING <110> Novo Nordisk A/S <120> Double-acylated GLP-1 derivatives <130> 8326.204-WO <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 31 <212> PRT <213> Homo sapiens <220> <221> mat_peptide <222> (1)..(31) <400> 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30

Claims (15)

Derivatives of GLP-1 analogs or pharmaceutically acceptable salts thereof; And pharmaceutically acceptable excipients, for use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome; Or as a pharmaceutical composition for delaying or preventing diabetes disease progression,
Derivatives of GLP-1 analogs are of the formula:
N ^ε27- [2- [2- [2-[[2- [2- [2-[[(4S) -4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl ] Amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl], N ^ε36- [2- [2- [2-[[2- [2- [2-[[(4S)-] 4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]-[Glu ²² , Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Arg ³⁴ , Lys ³⁶ ] -GLP-1- (7-37) -peptidyl-Glu-Gly

Having a pharmaceutical composition. Derivatives of GLP-1 analogs or pharmaceutically acceptable salts thereof; And pharmaceutically acceptable excipients, for use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome; Or as a pharmaceutical composition for delaying or preventing diabetes disease progression,
Derivatives of GLP-1 analogs are of the formula:
N ^ε27- [2- [2- [2-[[2- [2- [2-[[(4S) -4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl ] Amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl], N ^ε36- [2- [2- [2-[[2- [2- [2-[[(4S)-] 4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]-[Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Arg ³⁴ , Lys ³⁶ ] -GLP-1- (7-37) -peptidyl-Glu

Having a pharmaceutical composition. Derivatives of GLP-1 analogs or pharmaceutically acceptable salts thereof; And pharmaceutically acceptable excipients, for use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome; Or as a pharmaceutical composition for delaying or preventing diabetes disease progression,
Derivatives of GLP-1 analogs are of the formula:
N ^α (N ^ε27- [2- [2- [2-[[2- [2- [2-[[(4S) -4-carboxy-4- [10- (4-carboxyphenoxy)) decanoylamino ] Butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl], N ^ε36- [2- [2- [2-[[2- [2- [2-[[( 4S) -4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]-[Val ²⁵ , Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Arg ³⁴ , Lys ³⁶ ] -GLP-1- (7-37) -peptidyl) -Gln

Having a pharmaceutical composition. Derivatives of GLP-1 analogs or pharmaceutically acceptable salts thereof; And pharmaceutically acceptable excipients, for use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome; Or as a pharmaceutical composition for delaying or preventing diabetes disease progression,
Derivatives of GLP-1 analogs are of the formula:
N ^ε27- [2- [2- [2-[[2- [2- [2-[[(4S) -4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl ] Amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl], N ^ε36- [2- [2- [2-[[2- [2- [2-[[(4S)-] 4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]-[Val ²⁵ , Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Gln ³⁴ , Lys ³⁶ ] -GLP-1- (7-37) -peptidyl-Glu

Having a pharmaceutical composition. Derivatives of GLP-1 analogs or pharmaceutically acceptable salts thereof; And pharmaceutically acceptable excipients, for use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome; Or as a pharmaceutical composition for delaying or preventing diabetes disease progression,
Derivatives of GLP-1 analogs are of the formula:
N ^ε27- [2- [2- [2-[[2- [2- [2-[[(4S) -4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl ] Amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl], N ^ε36- [2- [2- [2-[[2- [2- [2-[[(4S)-] 4-carboxy-4- [10- (4-carboxyphenoxy) decanoylamino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]-[Aib ⁸ , Glu ²² , Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Arg ³⁴ , Lys ³⁶ ] -GLP-1- (7-37) -peptidyl-Glu-Gly

Having a pharmaceutical composition. Derivatives of GLP-1 analogs or pharmaceutically acceptable salts thereof; And pharmaceutically acceptable excipients, for use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome; Or as a pharmaceutical composition for delaying or preventing diabetes disease progression,
Derivatives of GLP-1 analogs are of the formula:
N ^ε27- [2- [2- [2-[[2- [2- [2-[[(4S) -4-carboxy-4- [12- (4-carboxyphenoxy) dodecanoylamino] buta Noyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl], N ^ε36- [2- [2- [2-[[2- [2- [2-[[(4S)) -4-carboxy-4- [12- (4-carboxyphenoxy) dodecanoylamino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]-[Glu ²² , Arg ²⁶ , Lys ²⁷ , Glu ³⁰ , Arg ³⁴ , Lys ³⁶ ] -GLP-1- (7-37) -peptidyl-Glu-Gly

Having a pharmaceutical composition. For use in the treatment or prevention of diabetes, obesity, cardiovascular disease, gastrointestinal disease, diabetic complications, or polycystic ovary syndrome, comprising the pharmaceutical composition of claim 1; Or a drug to delay or prevent the progression of diabetes disease. delete delete delete delete delete delete delete delete