Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/26—Glucagons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons

Definitions

• GLP-1 Glucagon-Like Peptide 1
• GLP-1 is a 37 amino acid peptide that is secreted by the L-cells of the intestine in response to food ingestion. It has been found to stimulate insulin secretion (insulinotropic action) , thereby causing glucose uptake by cells and decreased serum glucose levels (see, e g., Mojsov, S., Int . J. Peptide Protein Research, £0:333-343 (1992)).
• GLP-1 (1-37) is poorly active and attention has been focused on truncated analogs, referred to as GLP compounds, which are biologically much more potent than GLP-1.
• GLP-1 (7-37) examples include GLP-1 (7-37), GLP- 1(7-36)NH 2 , Gly 8 -GLP-l(7-37)OH and Ser 3 -GLP-1 (7-37) OH. Because of their ability to stimulate insulin secretion, GLP compounds show great promise as agents for the treatment of diabetes, obesity, and related conditions.
• GLP-1 compounds can exist in at least two different forms.
• the first form is physiologically active and dissolves readily in aqueous solution at physiological pH (7.4).
• the second form has little or no insulinotropic activity and is substantially insoluble in water at pH 7.4.
• the inactive form is readily produced when aqueous GLP-1 solutions are agitated, exposed to hydrophobic surfaces or have large air/water interfaces.
• the tendency to convert to the insoluble form considerably complicates the production of commercial quantities of active GLP-1 compounds; mixing operations or continuous movement through a pump are common operations in bulk manufacturing processes and these operations cause the agitation, air/water interfaces and/or contact with hydrophobic surfaces that results in the insoluble form. Conversion to the inactive form may also occur during storage or after administration to a patient, further complicating the use of these compounds as drugs.
• GLP-1 analogs with modifications at one or more of the following positions: 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36 or 37, show markedly decreased propensity to aggregate compared with GLP-1 (7-37) OH.
• Many of these analogs retain GLP-1 receptor activation that is comparable and in some cases greater than known GLP- 1 compounds such as GLP-1 (7-37) OH and Val 8 -GLP-1 (7-37) OH.
• the aggregation time of Val 8 -Glu 22 -GLP (7-37) OH is over twenty fold greater and its GLP-1 receptor activation is about 25% greater than GLP-1 (7-37) OH.
• One embodiment of the present invention is a polypeptide having the amino acid sequence of formula I (SEQ ID NO: 1) : His-Xaa 8 -Glu-Gly-Xaa ⁇ -Xaa ⁇ 2 -Thr-Ser-Asp-Xaai 6 -Ser- Ser-Tyr-Leu-Glu-Xaa 22 -Xaa 3 -Xaa 24 -Ala-Xaa 2 6 ⁇ Xaa 27 -Phe- Ile-Ala-Xaa 3 ⁇ -Leu-Xaa 33 -Xaa 34 -Xaa 3 5 ⁇ Xaa 3 6-R formula I (SEQ ID NO: 1)
• Xaae is: Gly, Ala, Val, Leu, He, Ser, or Thr;
• Xaan is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
• Xaa ⁇ 2 is: His, Trp, Phe, or Tyr;
• Xaai 6 is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala;
• Xaa 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, Cys, or Cysteic
• Xaa 23 is: His, Asp, Lys, Glu, or Gin
• Xaa 2 is: Glu, His, Ala, or Lys
• Xaa 2 6 is: Asp, Lys, Glu, or His;
• Xaa 2 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys
• Xaa 3o is: Ala, Glu, Asp, Ser, or His
• Xaa 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu
• Xaa 34 is: Glu, Lys, or Asp;
• Xaa 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro,
• Xaa 36 is: Arg, Glu, or His;
• R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH 2 , Gly, Gly-Pro, or Gly-Pro-NH 2 , or is deleted.
• polypeptide does not have the sequence of GLP-1 (7-37) OH or GLP-1 (7-36) -NH 2 and provided that the polypeptide is not Gly 8 -GLP-1 (7-37) OH, Gly 8 -GLP- 1(7-36)NH 2 , Val 8 -GLP-1 (7-37) OH, Val 8 -GLP-1 (7-36) NH 2 , Leu 8 -
• GLP-l(7-37)OH Leu 8 -GLP-1 (7-36) NH 2 , Ile 8 -GLP-1 (7-37 ) OH, Ile 8 - GLP-1(7-36)NH 2 , Ser 8 -GLP-1 (7-37) OH, Ser 8 -GLP-1 (7-36) NH 2 , Thr 8 -GLP-l(7-37)OH, or Thr 8 -GLP-1 (7-36) NH 2 , Ala n -Glp-1 (7- 37)OH, Ala n -Glp-1(7-36)NH 2 , Ala 16 -Glp-1 (7-37) OH, Ala 16 -Glp- 1(7-36)NH 2 , Ala 27 -Glp-l(7-37)OH, Ala 27 -Glp-1 (7-36) NH 2 , Glu 27 - Glp-l(7-37)OH, Glu 7 -Glp
• Another embodiment of the present invention is a polypeptide having the amino acid sequence of formula II (SEQ ID NO: 2) :
• Xaas is: Gly, Ala, Val, Leu, He, Ser, or Thr;
• Xaa ⁇ 2 is: His, Trp, Phe, or Tyr;
• Xaai ⁇ is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala;
• Xaa 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, Cys, or Cysteic Acid
• Xaa 23 is: His, Asp, Lys, Glu, or Gin;
• Xaa26 is: Asp, Lys, Glu, or His;
• Xaa 3 o is: Ala, Glu, Asp, Ser, or His;
• Xaa 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His, or Glu; R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH 2 , Gly, Gly-Pro, or Gly-Pro-NH 2 , or is deleted.
• polypeptide does not have the sequence of - GLP-1 (7-37) OH or GLP-1 (7-36) -NH 2 and provided that the polypeptide is not Gly 8 -GLP-1 (7-37) OH, Gly 8 -GLP-1 (7-36) H 2 , Val 8 -GLP-l(7-37)OH, Val 8 -GLP-1 (7-36) NH 2 , Leu 8 -GLP-1 (7-37) OH, Leu 8 -GLP-1(7-36)NH 2 , Ile 8 -GLP-1 (7-37) OH, Ile 8 -GLP-1 (7-36) NH 2 , Ser 8 -GLP-l(7-37)OH, Ser 8 -GLP-1 (7-36) NH 2 , Thr 8 -GLP-1 (7-37) OH, Thr 8 -GLP-1 (7-36) NH 2 , Ala 16 -GLP (7-37) OH, or Ala 16 -Gl
• Xaa 8 is: Gly, Ala, Val, Leu, He, Ser, or Thr;
• Xaa 22 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, Cys, or Cysteic
• Xaa 23 is: His, Asp, Lys, Glu, or Gin
• Xaa 2 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys
• Xaa 3 o is: Ala, Glu, Asp, Ser, or His;
• R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH 2 , Gly, Gly-Pro, or Gly-Pro-NH 2 , or is deleted.
• polypeptide does not have the sequence of GLP-1 (7-37)OH or GLP-1 (7-36) -NH 2 and provided that the polypeptide is not Gly 8 -GLP-1 (7-37) OH, Gly 8 -GLP- 1(7-36)NH 2 , Val 8 -GLP-l(7-37)OH, Val 8 -GLP-1 (7-36) NH 2 , Leu 8 - GLP-l(7-37)OH, Leu 8 -GLP-1 (7-36) NH 2 , Ile 8 -GLP-1 (7-37) OH, Ile 8 - GLP-1 (7-36) NH 2 , Ser 8 -GLP-1 (7-37) OH, Ser 8 -GLP-1 (7-36) NH 2 ,
• Another embodiment of the present invention is a polypeptide having the amino acid sequence of formula IV (SEQ ID NO: 4) :
• Xaa is L-histidine, D-histidine, desamino-histidine, 2amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ - fluoromethyl-histidine or ⁇ -methyl-histidine;
• Xaa 8 is glycine, alanine, valine, leucine, isoleucine, serine or threonine.
• Xaa 8 is glycine, valine, leucine, isoleucine, serine or threonine;
• Xaa 2 is aspartic acid, glutamic acid, glutamine, asparagine, lysine, arginine, cysteine, or cysteic acid.
• R is -NH 2 or Gly (OH) .
• GLP-1 glucagon-like peptide-1
• Another embodiment of the present invention is a method of stimulating the GLP-1 receptor in a subject in need of GLP-1 receptor stimulation.
• the method comprises the step of administering to the subject an effective amount of the GLP-1 compounds described herein or the polypeptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4.
• Yet another embodiment of the present invention is the GLP-1 compounds described herein or the polypeptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 for use in stimulating the GLP-1 receptor in a subject in need of GLP-1 receptor stimulation.
• the GLP-1 compounds of the present invention retain GLP-1 receptor activation ability and, in addition, have decreased propensity to aggregate compared with other GLP-1 compounds. As a result, solutions of these compounds can be agitated with minimal conversion to the insoluble, inactive form. This advantage greatly simplifies the manufacturing process. In addition, it is expected that little or no in vivo aggregation will occur after administration to patients, thereby increasing activity and minimizing the potential for adverse reactions. In addition, these GLP-1 compounds are resistant to diaminopeptidase IV degradation and bind zinc and are therefore believed to provide extended time action in vivo.
• Figure 1 shows the amino acid sequences of Val 8 -Glu 22 - GLP-l(7-37)OH (SEQ ID NO: 5), Val 8 -Asp 22 -GLP-1 (7-37) OH (SEQ ID NO: 6), Val 8 -Arg 22 -GLP-1 (7-37) OH (SEQ ID NO: 7) and Val 8 - Lys 22 -GLP-l(7-37)OH (SEQ ID NO: 8).
• Figure 2 shows the amino acid sequences of Gly 8 -Glu 22 - GLP-l(7-37)OH (SEQ ID NO: 9), Gly 8 -Asp 22 -GLP-1 (7-37 ) OH (SEQ ID NO: 10), Gly 8 -Arg 22 -GLP-l(7-37)OH (SEQ ID NO: 11) and Gly 8 -Lys 22 -GLP-l(7-37)OH (SEQ ID NO: 12).
• Figure 3 shows the amino acid sequence of Val 8 -Glu 30 - GLP-l(7-37)OH (SEQ ID NO: 13), Gly 8 -Glu 30 -GLP-1 (7-37 ) OH (SEQ ID NO: 14), Val 8 -His 37 -GLP-1 (7-37) OH (SEQ ID NO: 15), and Gly 8 -His 37 -GLP-l(7-37)OH (SEQ ID NO: 16).
• Figure 4 shows the amino acid sequence of Val 8 -Glu 22 - Ala 27 -GLP-l(7-37)OH (SEQ ID NO: 17) and Val 8 -Lys 22 -Glu 23 -GLP- l(7-37)OH (SEQ ID NO: 18) .
• a GLP-1 compound is a polypeptide having from about twenty-five to about thirty-nine naturally occurring or non- naturally occurring amino acids and has sufficient homology to GLP-1 (7-37)OH such that it exhibits insulinotropic activity.
• non-naturally occurring amino acids include ⁇ -methyl amino acids (e.g., ⁇ -methyl alanine), D- amino acids, histidine-like amino acids (e.g., 2-amino- histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ - fluoromethyl-histidine and ⁇ -methyl-histidine) , amino acids having an extra methylene in the side chain (“homo" amino acids) and amino acids in which a carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid).
• the GLP-1 compounds of the present invention comprise only naturally occurring amino acids except as otherwise specifically provided herein.
• a GLP-1 compound typically comprises a polypeptide having the amino acid sequence of GLP-1 (7-37) OH, an analog of GLP-1 (7-37) OH, a fragment of GLP-1 (7-37) OH or a fragment of a GLP-1 (7-37) OH analog.
• GLP-1 (7-37) OH has the amino acid sequence of SEQ ID NO: 19:
• GLP-1 (7-37) OH has been assigned number residue 7 and the carboxy-terminus, number 37.
• the other amino acids in the polypeptide are numbered consecutively, as shown in SEQ ID NO: 19.
• position 12 is phenylalanine and position 22 is glycine.
• the C-terminal is in the traditional carboxyl form.
• a "GLP-1 fragment” is a polypeptide obtained, after truncation of one or more amino acids from the N-terminus and/or C-terminus of GLP-1 (7-37) OH or a GLP-1 (7-37) OH analog.
• the nomenclature used to describe GLP-1 (7-37) OH carries over to GLP-1 fragments.
• GLP-1 (9-36) OH denotes a GLP-1 fragment obtained by truncating two amino acids from the N-terminus and one amino acid from the C- terminus .
• the amino acids in the fragment are denoted by the same number as the corresponding amino acid in GLP-1 (7- 37) OH.
• the N-terminal glutamic acid in GLP- l(9-36)OH is at position 9; position 12 is occupied by phenylalanine; and position 22 is occupied by glycine, as in GLP-1 (7-37)OH.
• GLP-1 compound also includes polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of GLP-1 (7-37) OH or fragments thereof.
• GLP-1 compounds of this type have up to about thirty-nine amino acids.
• the amino acids in the "extended" GLP-1 compound are denoted by the same number as the corresponding amino acid in GLP-1 (7-37) OH.
• the N-terminus amino acid of a GLP-1 compound obtained by adding two amino acids to the N-terminal of GLP-1 (7-37) OH is at position 5; and the C-terminus amino acid of a GLP-1 compound obtained by adding one amino acids to the C-terminal of GLP-1 (7-37) OH is at position 38.
• position 12 is occupied by phenylalanine and position 22 is occupied by glycine in both of these "extended" GLP-1 compounds, as in GLP-1 (7-37) OH.
• Amino acids 1-6 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of GLP-1 (1-37) OH.
• Amino acids 38-45 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of glucagon or exendin-4.
• a "GLP-1 analog” has sufficient homology to GLP-1 (7- 37) OH or a fragment of GLP-1 (7-37) OH such that the analog has insulinotropic activity.
• a GLP-1 analog has the amino acid sequence of GLP-1 (7-37) OH or a fragment thereof, modified so that from one, two, three, four or five amino acids differ from the amino acid in corresponding position of GLP-1 (7-37) OH or the fragment of GLP-1 (7-37) OH.
• the substituting amino acid and its position is indicated prior to the parent structure.
• Glu 22 -GLP-1 (7-37) OH designates a GLP-1 compound in which the glycine normally found at position 22 of GLP-1 (7-37) OH has been replaced with glutamic acid
• Val 8 -Glu 22_ GLP-l (7-37) OH designates a GLP-1 compound in which alanine normally found at position 8 and glycine normally found at position 22 of GLP-1 (7-37) OH have been replaced with valine and glutamic acid, respectively.
• the N-terminus of a GLP-1 compound is generally unsubstituted but can also be alkylated or acylated (preferably C1-C20) .
• the C-terminus can be unsubstituted, as is the case with GLP-1 (7-37) OH, amidated with - ⁇ H 2 , -NHR or NRR' or esterified with -OR".
• R and R' are independently alkyl or acyl groups (preferably C1-C20) .
• R" is an alkyl (C1-C20) .
• GLP-1 (7-36) NH 2 is an example of an "amidated GLP compound".
• the GLP-1 compounds of the present invention have a C-terminus that is unsubstituted or substituted with -NH 2 .
• GLP-1 compounds of the present invention comprise GLP-1 analogs or fragments of GLP-1 analogs wherein the backbone for such analogs or fragments contains an amino acid other than alanine at position 8 (position 8 analogs) .
• the backbone may also include L-histidine, D- histidine, or modified forms of histidine such as desamino- histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine, or ⁇ -methyl- histidine at position 7.
• these position 8 analogs contain one or more additional changes at positions 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36, and 37 compared to the corresponding amino acid of native GLP-1 (7-37) OH. It is more preferable that these position 8 analogs contain one or more additional changes at positions 12, 16, 22, 23, 26, 30, 35, and 37 compared to the corresponding amino acid of native GLP-1 (7-37) OH. It is even more preferable that these position 8 analogs contain one or more additional changes at positions 22, 23, 27, 30, and 37 compared to the corresponding amino acid of native GLP-1 (7-37)OH.
• these analogs have 6 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH. More preferred analogs have 5 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH or have 4 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH. It is even more preferable that these analogs have 3 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH. It is most preferable that these analogs have 2 or fewer changes compared to the corresponding amino acids in native GLP-1 (7-37) OH.
• the GLP-1 compounds of the present invention generally aggregate at least about 5 times less rapidly than GLP-1 (7-37) OH when assessed, for example, by the aggregation assay described in Example 3, preferably at least 20 times less rapidly, more preferably at least 40 times less rapidly, more preferably at least about 50 times less rapidly, even more preferably about 60 times less rapidly, and even more preferably at least about 65 times less rapidly.
• GLP-1 compounds described herein are analogs of GLP-1 (7-36) NH 2 or GLP-1 (7-37 ) OH.
• the amino acid at position 22 of the GLP-1 compound of the present invention has a side chain which comprises at least two carbon atoms and a polar or charged functional group.
• Aspartic acid which has a methylene and carboxyl carbon
• the side chain of the amino acid at position 22 is a straight or branched chain alkyl group with from two to six carbon atoms and a charged functional group, e.g., a carboxylic acid, an amine, guanidino group or a sulfonic acid group.
• examples of preferred amino acids at position 22 include glutamic acid, aspartic acid, arginine and lysine.
• position 8 is preferably glycine, valine, leucine, isolecine, serine, threonine or methionine and more preferably valine or glycine.
• An example of an amino acid with a sulfonic acid group in the side chain cysteic acid (-NH-CH (CH 2 S0 3 ) -CO-, abbreviated as "Cya") .
• position 8 is preferably glycine, valine, leucine, isolecine, serine, threonine or methionine and more preferably valine or glycine.
• the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 30 is glutamic acid, aspartic acid, serine, or histidine and more preferably glutamatic acid.
• the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 37 is histidine, lysine, arginine, threonine, serine, glutamic acid, aspartic acid, tryptophan, tyrosine, phenylalanine and more preferably histidine.
• the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 22 is glutamic acid, lysine, aspartic acid, or arginine and more preferably glutamine acid or lysine and position 23 is lysine, arginine, glutamic acid, aspartic acid, and histidine and more preferably lysine or glutamic acid.
• the amino acid at position 8 is preferably glycine, valine, leucine, isoleucine, serine, threonine, or methionine and more preferably valine or glycine and position 22 is glutamic acid, lysine, aspartic acid, or arginine and more preferably glutamine acid or lysine and position 27 is alanine, lysine, arginine, tryptophan, tyrosine, phenylalanine, or histidine and more preferably alanine.
• the GLP-1 compounds of the present invention have an amino acid at position 8 and have one, two, or three amino acids selected from the group consisting of position 11, position 12, position 16, position 22, position 23, position 24, position 26, position 27, position 30, position 33, position 34, position 35, position 36, and position 37, which differ from the amino acid at the corresponding position of native GLP-1 (7-37) OH.
• the GLP-1 compounds of the present invention have one or two amino acids, in addition to the amino acid at position 8, selected from the group consisting of position 11, position 12, position 16, position 22, position 23, position 24, position 26, position 27, position 30, position 33, position 34, position 35, position 36, and position 37, which differ from the amino acid at the corresponding position of native .
• GLP-1 (7-37) OH is another preferred embodiment, selected from the group consisting of position 11, position 12, position 16, position 22, position 23, position 24, position 26, position 27, position 30, position 33, position 34, position 35, position 36, and position 37, which differ from the amino acid at the corresponding position of native .
• the GLP-1 compounds of the present invention can have amino acids in addition to those at position 8, 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36, and 37 which differ from the amino acid at the corresponding position of GLP-1 (7-37) or a fragment of GLP- 1(7-37).
• the amino acids other than those at positions 8, 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36, and 37 in the GLP compound which differ from the amino acid in corresponding position of GLP-1 (7-37) OH are preferably conservative substitutions and, more preferably, are highly conservative substitutions.
• the GLP-1 compounds of the present invention have zero, one, two or three amino acids in addition to the amino acids at positions 8 and 22 which differ from the amino acid at the corresponding position of GLP-1 (7-37) OH or a GLP-1 (7-37) OH fragment.
• one or more of the amino acids at positions 7, 21 and 27 of the GLP-1 compound differ from the corresponding amino acid in GLP-1 (7-37)OH or a GLP-1 (7-37 ) OH fragment, in addition to the amino acids at positions 8 and 22.
• positions 7, 8 and 22 differ from the amino acid at the corresponding position of GLP-1 (7-37) OH (or a fragment thereof) .
• GLP-1 (7-37) OH or a fragment thereof
• other improved GLP-1 compounds with reduced aggregating properties can be obtained from known, biologically active GLP-1 compounds by replacing glycine at position 22 and preferably alanine at position 8 -of these compounds with a suitable amino acid, as described herein.
• Known biologically active GLP-1 compounds are disclosed in U.S. Patent No 5,977,071 to Hoffmann, et al . , U.S. Patent No. 5,545,618 to Buckley, et al . , Adelhorst, et al . , J. Biol . Chem .
• a “conservative substitution” is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape.
• Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in the their side chains differs by no more than one.
• Amino acids with phenyl or substituted phenvl ⁇ roups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids.
• Group II glutamic acid, aspartic acid and non- naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point) .
• Group III lysine, ornithine, arginine and non- naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point) .
• Group IV glutamine, asparagine and non-naturally occurring amino acids with amide substituted C1-C4 aliphatic side chains (unbranched or one branch point) .
• Group V phenylalanine, phenylglycine, tyrosine and tryptophan.
• a “highly conservative substitution” is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape.
• Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have the same number of branches in the their side chains.
• Example of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine. Examples of substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.
• GLP-1 compound of the present invention is a polypeptide having the amino acid sequence of SEQ ID NO:l.
• the GLP-1 compound is GLP-1 (7-37) OH except that Xaa 8 is Gly or Val, Xaa 22 is Glu or Lys, and Xaa 23 is Glu or Lys.
• the GLP-1 compound is GLP-1 (7-37) OH except that Xaa 8 is Gly or Val and Xaa 3 o is Glu.
• An additional example is a GLP-1 compound which is GLP-1 (7-37) OH except that Xaa 8 is Gly or Val and Xaa 37 is His.
• GLP-1 compound of the present invention is a polypeptide having the amino acid sequence of SEQ ID. No. 4.
• Xaa is L-histidine, D-histidine, desamino-histidine, 2amino-histidine, ⁇ - hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine and ⁇ -methyl-histidine
• Xaa 8 is glycine, alanine, valine, leucine, isoleucine, serine, or threonine, and preferably, glycine, valine, leucine, isoleucine, serine, or threonine
• R is -NH 2 or Gly (OH) and Xaa2 2 is lysine, glutamic acid, aspartic acid or arginine in SEQ ID NO: 4.
• Xaa 7 is L-histidine
• Xaa 8 is glycine or valine
• Xaa2 2 is lysine
• glutamic acid aspartic acid or arginine
• R is Gly (OH).
• Xaa 7 , Xaa 8 and R in SEQ ID NO: 4 are as described above and Xaa 22 is an amino acid with a side chain comprising a sulfonic acid group, e.g., cysteic acid.
• the amino acid at position 8 is not a D-amino acid and does not have the side chain of glycine, serine, threonine, cysteine or beta-alanine when the amino acid at position 22 has a C1-C2 alkyl side chain, a C1-C2 hydoxylated alkyl side chain or a C1-C2 thiolated alkyl chain (e.g., cysteine).
• the amino acid at position 8 is not a D-amino acid and does not have the side chain of glycine, serine, threonine, cysteine or beta- alanine when the amino acid at position 22 has a C1-C4 alkyl side chain, a C1-C4 hydroxylated alkyl side chain or a C1-C4 thiolated alkyl chain.
• the amino acid at position 8 is glycine, valine, leucine, isoleucine, methionine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine, phenylalanine, tyrosine, histidine or tryptophan; and the amino acid at position 22 is aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine or histidine.
• GLP-1 compounds of the present invention include Glu 22 -GLP-1 (7-37) OH (SEQ ID NO: 20),
• GLP-1 compound also includes pharmaceutically acceptable salts of the compounds described herein.
• a GLP-1 compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt.
• Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl- sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like
• organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl- sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
• salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, ethoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate
• Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
• bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.
• the GLP-1 compounds can be used to treat subjects with a wide variety of diseases and conditions. It is believed that GLP-1 compounds, including those of the present invention, exert their biological effects by acting at a receptor referred to as the "GLP-1 receptor" (see U.S. Patent No. 5,670,360 to Thorrens) . Subjects with diseases and/or conditions that respond favorably to GLP-1 receptor stimulation or to the adminstration of GLP-1 compounds can therefore be treated with the GLP-1 compounds of the present invention. These subjects are said to "be in need of treatment with GLP-1 compounds" or "in need of GLP-1 receptor stimulation".
• Non- insulin dependent diabetes insulin dependent diabetes
• stroke see WO 00/16797 by Efendic
• myocardial infarction see WO 98/08531 by Efendic
• obesity see WO 98/19698 by Efendic
• catabolic changes after surgery see U.S. Patent No. 6,006,753 to Efendic
• functional dyspepsia see WO 99/64060 by Efendic
• subjects requiring prophylactic treatment with a GLP-1 compound e.g., subjects at risk for developing non- insulin dependent diabetes (see WO 00/07617) .
• Subjects with impaired glucose tolerance or impaired fasting glucose subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects with a partial pancreatecto y, subjects having one or more parents with non-insulin dependent diabetes, subjects who have had gestational diabetes and subjects who have had acute or chronic pancreatitis are at risk for developing non-insulin dependent diabetes.
• an “effective amount” of a GLP-1 compound is the quantity which results in a desired therapeutic and/or prophylactic effect without causing unacceptable side- effects when administered to a subject in need of GLP-1 receptor stimulation.
• a “desired therapeutic effect” includes one or more of the following: 1) an amelioration of the symptom (s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment.
• an "effective amount" of a GLP-1 compound for the treatment of diabetes is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy or kidney disease.
• An "effective amount” of a GLP-1 compound for the prevention of diabetes is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood giucose levels that require treatment with anti-hypoglycaemic drugs such as sulfonyl ureas, thiazolidinediones, insulin and/or bisguanidines.
• an "effective amount" of the GLP-1 compound administered to a subject will also depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
• a therapeutically effective amount of GLP-1 compound can range from about 0.01 mg per day to about 1000 mg per day for an adult.
• the dosage ranges from about 0.1 mg per day to about 100 mg per day, more preferably from about 1.0 mg/day to about 10 mg/day.
• the GLP-1 compounds of the present invention can, for example, be administered orally, by nasal administration, inhalation or parenterally.
• Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.
• the GLP-1 compounds can be administered to the subject in conjunction with an acceptable pharmaceutical carrier, diluent or excipient as part of a pharmaceutical composition for treating the diseases discussed above.
• the pharmaceutical composition can be a solution or, if administered parenterally, a suspension of the GLP- 1 compound or a suspension of the GLP-1 compound complexed with a divalent metal cation, as described below.
• Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the peptide or peptide derivative.
• Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ l benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer 's- lactate and the like.
• suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol.
• a "subject” is a mammal, preferably a human, but can also be an animal, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
• companion animals e.g., dogs, cats, and the like
• farm animals e.g., cows, sheep, pigs, horses, and the like
• laboratory animals e.g., rats, mice, guinea pigs, and the like.
• the GLP-1 compounds of the present invention can be complexed with a suitable divalent metal cation.
• Divalent metal complexes of GLP-1 compounds are generally insoluble in aqueous solution around physiological pH. Thus, these complexes can be administered subcutaneously as suspensions and show a decreased rate of release in vivo, thereby extending the time action the compound.
• suitable divalent metal cations include Zn ++ , Mn ++ , Fe ++ , Ca ++ , Co ++ , Cd ++ , Ni ++ , and the like. Zn++ is preferred.
• a GLP-1 is dissolved in a suitable buffer and in the presence of a metal salt.
• the mixture is allowed to incubate at ambient temperature to allow the complex to precipitate.
• Suitable buffers are those which maintain the mixture at a pH range from about 3.0 to about 9.0 and do not interfere with the complexation reaction. Examples include phosphate buffers, acetate buffers, citrate buffers and Goode's buffers, e.g., HEPES, Tris and Tris acetate.
• Suitable metal salts are those in which the metal is available for complexation.
• suitable zinc salts include zinc chloride, zinc acetate, zinc oxide, and zinc sulfate.
• a divalent metal cationic salt such as zinc chloride is provided in excess to provide a molar ratio of up to about 50 molecules of a divalent metal cation for each molecule of GLP-1 compound.
• Insulinotropic activity refers to stimulating insulin secretion in response to elevated glucose levels, thereby causing glucose uptake by cells and decreased serum glucose levels. Insulinotropic activity can be assessed by methods known in the art, including using in vivo experiments and in vi tro assays that measure GLP-1 receptor binding activity or receptor activation, e.g., assays employing pancreatic islet cells or insulinoma cells, as described in EP 619,322 to Gelfand, et al . , and U.S. Patent No. 5,120,712, respectively. The entire teachings of these references are incorporated herein by reference.
• the GLP-1 compounds of the present invention can be prepared by using standard methods of solid-phase peptide synthesis techniques.
• Peptide synthesizers are commercially available from, for example, Applied Biosystems in Foster City CA. Reagents for solid phase synthesis are commercially available, for example, from Midwest Biotech (Fishers, IN) .
• Solid phase peptide synthesizers can be used according to manufacturers instructions for blocking interfering groups, protecting the amino acid to be reacted, coupling, decoupling, and capping of unreacted amino acids.
• an ⁇ -N-carbamoyl protected amino acid and the N-terminal amino acid on the growing peptide chain on a resin is coupled at room temperature in an inert solvent such as dimethylformamide, ⁇ -methylpyrrolidone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole and a base such as diisopropylethylamine.
• the ⁇ -N-carbamoyl protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N- protected amino acid to be added to the peptide chain.
• Suitable amine protecting groups are well known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991, the entire teachings of which are incorporated by reference. Examples include t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) .
• the peptides are also synthesized using standard automated solid-phase synthesis protocols using t- butoxycarbonyl- or fluorenylmethoxycarbonyl-alpha-amino acids with appropriate side-ghain protection. After completion of synthesis, peptides are cleaved from the solid-phase support with simultaneous side-chain deprotection using standard hydrogen fluoride methods. Crude peptides are then further purified using Reversed- Phase Chromatography on Vydac C18 columns using acetonitrile gradients in 0.1% trifluoroacetic acid (TFA) . To remove acetonitrile, peptides are lyophilized from a solution containing 0.1 % TFA, acetonitrile and water. Purity can be verified by analytical reversed phase chromatography. Identity of peptides can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers at neutral pH.
• Boc Gly- PAM resin was placed in a standard 60 ml reaction vessel and double couplings were run on an Applied Biosytems ABI430A peptide synthesizer.
• the following side-chain protected amino acids (2 mmole cartridges of Boc amino acids) were obtained from Midwest Biotech (Fishers, IN) and used in the synthesis:
• Arg-Tosyl Asp- ⁇ -cyclohexyl ester (CHXL), Glu- ⁇ - cycohexyl ester (CHXL), His-benzyloxymethyl (BOM) , Lys-2- chlorobenzyloxycarbonyl (2C1-Z), Met-sulfoxide (0), Ser-O- benzyl ether (OBzl) , Thr-O-benzyl ether (OBzl), Trp-formyl (CHO) and Tyr-2-bromobenzyloxycarbonyl (2Br-Z) and Boc Gly PAM resin.
• Trifluoroacetic acid (TFA), di- isopropylethylamine (DIEA) , 0.5 M hydroxybenzotriazole (HOBt) in DMF and 0.5 M dicyclohexylcarbodiimide (DCC) in dichloromethane were purchased from PE-Applied Biosystems (Foster City, CA) .
• Dimethylformamide (DMF-Burdick and Jackson) and dichloromethane (DCM-Mallinkrodt) were purchased from Mays Chemical Co. (Indianapolis, IN) . Standard double couplings were run using either symmetric anhydride or HOBt esters, both formed using DCC.
• FMOC Gly WANG resin purchased from NovaBiochem, LaJolla, CA
• Approximately 114 mg (50 mMole) FMOC Gly WANG resin purchased from NovaBiochem, LaJolla, CA
• Analogs with a C-terminal amide were prepared using 75 mg (50 ⁇ mole) Rink Amide AM resin (NovaBiochem, LaJolla, CA) .
• the following FMOC amino acids were purchased from
• HOBt Hydroxybenzotrizole
• DIC di-isopropylcarbodiimde
• DIEA di-isopropylethylamine
• Pip piperidine
• Reagent K 0.75 g phenol, 0.5 ml thioanisole, 0.25 ml ethanedithiol, 0.5 ml water per 10 ml trifluoroacetic acid (TFA) , all purchased from Aldrich Chemical Co., Milwaukee, WI] .
• TFA trifluoroacetic acid
• the TFA filtrates were added to 40 ml ethyl ether and the precipitants centrifuged 2 minutes at 2000 rpm. The supernatants were decanted, the pellets re-suspended in 40 ml ether, re-centrifuged, re- decanted, dried under nitrogen and then in vacuo.
• GLP peptides of this invention were analyzed with respect to their potential to aggregate in solution.
• peptides in solution were stirred at elevated temperature in a suitable buffer while recording turbidity at 350 nm as a function of time. Time to the onset of aggregation was measured to quantify the potential of a given GLP molecule to aggregate under these stressed conditions.
• a GLP-1 compound was first dissolved under alkaline conditions (pH 10.5) for 30 minutes to dissolve any pre- aggregated material. The solution was then adjusted to pH 7.4 and filtered. Specifically, 4 mg of a lyophilized GLP-1 compound was dissolved in 3 ml of 10 mM phosphate/10 mM citrate. The pH was adjusted to 10.0-10.5 and held for 30 minutes. The solution was adjusted with HC1 to pH 7.4 and filtered through a suitable filter, for example a Millex GV syringe filter (Millipore Corporation, Bedford, MA) .
• a suitable filter for example a Millex GV syringe filter (Millipore Corporation, Bedford, MA) .
• This solution was then diluted to a final sample containing 0.3 mg/mL protein in 10 mM citrate, 10 mM phosphate, 150 mM NaCl, and adjusted to pH 7.4 to 7.5.
• the sample was incubated at 37°C in a quartz cuvette. Every five minutes the turbidity of the solution was measured at 350 nm on an AVIV Model 14DS UV-VIS spectrophotometer (Lakewood, NJ) . For 30 seconds prior to and during the measurement the solution was stirred using a magnetic stir bar from Starna Cells, Inc. (Atascadero, CA) . An increase in OD at 350 nm indicates aggregation of the GLP-peptide.
• the cuvette was cleaned between experiments with a caustic soap solution (e.g., Contrad-70) .
• a caustic soap solution e.g., Contrad-70
• the results for a number of GLP-1 compounds of the present invention are reported in Table 1 as the time in hours required for the compound to aggregate. As can be seen, the compounds of the present invention show greatly increased aggregation times over GLP-1 compounds known in the prior art.
• GLP-1 Compound Time in Hours Activation
• Example 5 Zinc Precipitation of GLP-1 Compounds Individual GLP-1 compounds were prepared as described in Examples 1 or 2. 3 mg of an individual lyophilized GLP molecule was dissolved in 3 ml 0.1 M HEPES buffer pH 10.5. The pH of the resulting solution was then adjusted to between 10.0 and 10.5 with 0.2 N NaOH. The solution was stirred at ambient temperature for 30 minutes and the solution was then adjusted to a pH of 7. with 0.2 N HC1.
• the solution was filtered through an appropriate syringe filter, for example a Millex GV syringe filter (Millipore Corporation, Bedford, MA) , and the concentration of the GLP- 1 compound was estimated by measuring the absorption at 280 nm in a spectrophotometer, for example a Beckman DU640.
• the protein concentration was then adjusted to 200 ⁇ M in HEPES pH 7.4.
• the filtered GLP-1 solutions (100 ⁇ l) were diluted with 100 ⁇ l of 0.1 M HEPES pH 7.4 containing various levels of zinc chloride in an ELISA plate, (e.g. Falcon Microt . estTM 96) resulting in 200 ⁇ l of solution containing various levels of zinc chloride and 100 ⁇ M GLP-1 compounds. These solutions were incubated at ambient temperature (22° C) for 18 hours and then centrifuged, for example, in a Jouan CR412 centrifuge with microplate adapters. 150 ⁇ l of the supernatants after centrifugation were then transferred to a UV-readable ELISA microtiter plate (e.g.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Diabetes (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Endocrinology (AREA)
• Gastroenterology & Hepatology (AREA)
• Zoology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Toxicology (AREA)
• Hematology (AREA)
• Obesity (AREA)
• Emergency Medicine (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Child & Adolescent Psychology (AREA)
• Urology & Nephrology (AREA)
• Heart & Thoracic Surgery (AREA)
• Vascular Medicine (AREA)
• Cardiology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (26)

Applications Claiming Priority (4)

Related Child Applications (2)

Publications (2)

Family

ID=26906843

Family Applications (1)

Country Status (32)

Cited By (60)

Families Citing this family (102)

Family Cites Families (17)

• 2001
  • 2001-06-01 BR BR0111562-6A patent/BR0111562A/pt not_active IP Right Cessation
  • 2001-06-01 AU AU2001264791A patent/AU2001264791B2/en not_active Ceased
  • 2001-06-01 EP EP01939252A patent/EP1294757B1/en not_active Expired - Lifetime
  • 2001-06-01 KR KR1020027017141A patent/KR100847615B1/ko not_active Expired - Fee Related
  • 2001-06-01 DE DE60137876T patent/DE60137876D1/de not_active Expired - Lifetime
  • 2001-06-01 DZ DZ013388A patent/DZ3388A1/xx active
  • 2001-06-01 MX MXPA02012203A patent/MXPA02012203A/es active IP Right Grant
  • 2001-06-01 PT PT01939252T patent/PT1294757E/pt unknown
  • 2001-06-01 AT AT06114553T patent/ATE424413T1/de active
  • 2001-06-01 SI SI200130915T patent/SI1695983T1/sl unknown
  • 2001-06-01 CN CNB018112137A patent/CN100469791C/zh not_active Expired - Fee Related
  • 2001-06-01 CA CA2412004A patent/CA2412004C/en not_active Expired - Fee Related
  • 2001-06-01 DK DK01939252T patent/DK1294757T3/da active
  • 2001-06-01 DE DE60124710T patent/DE60124710T2/de not_active Expired - Lifetime
  • 2001-06-01 NZ NZ522330A patent/NZ522330A/en unknown
  • 2001-06-01 EP EP06114553A patent/EP1695983B1/en not_active Expired - Lifetime
  • 2001-06-01 ES ES06114553T patent/ES2321439T3/es not_active Expired - Lifetime
  • 2001-06-01 US US10/276,772 patent/US7084243B2/en not_active Expired - Fee Related
  • 2001-06-01 PL PL360406A patent/PL206302B1/pl not_active IP Right Cessation
  • 2001-06-01 PT PT06114553T patent/PT1695983E/pt unknown
  • 2001-06-01 AU AU6479101A patent/AU6479101A/xx active Pending
  • 2001-06-01 JP JP2002504286A patent/JP4716641B2/ja not_active Expired - Fee Related
  • 2001-06-01 CZ CZ20024088A patent/CZ304002B6/cs not_active IP Right Cessation
  • 2001-06-01 EA EA200300026A patent/EA008837B1/ru not_active IP Right Cessation
  • 2001-06-01 WO PCT/US2001/016474 patent/WO2001098331A2/en not_active Ceased
  • 2001-06-01 HU HU0301212D patent/HU229208B1/hu unknown
  • 2001-06-01 HU HU0301212A patent/HU229108B1/hu not_active IP Right Cessation
  • 2001-06-01 DK DK06114553T patent/DK1695983T3/da active
  • 2001-06-01 IL IL15345301A patent/IL153453A0/xx unknown
  • 2001-06-01 HR HR20020996A patent/HRP20020996B1/xx not_active IP Right Cessation
  • 2001-06-01 SK SK1778-2002A patent/SK287757B6/sk not_active IP Right Cessation
  • 2001-06-01 AT AT01939252T patent/ATE346093T1/de active
  • 2001-06-01 ES ES01939252T patent/ES2275685T3/es not_active Expired - Lifetime
  • 2001-06-11 TW TW090114055A patent/TWI321134B/zh not_active IP Right Cessation
  • 2001-06-12 AR ARP010102775A patent/AR031701A1/es active IP Right Grant
  • 2001-06-13 EG EG20010628A patent/EG24755A/xx active
  • 2001-06-14 MY MYPI20012806A patent/MY137350A/en unknown
  • 2001-06-15 PE PE2001000583A patent/PE20011363A1/es not_active Application Discontinuation
• 2002
  • 2002-12-11 NO NO20025949A patent/NO330686B1/no not_active IP Right Cessation
  • 2002-12-15 IL IL153453A patent/IL153453A/en active IP Right Grant
• 2006
  • 2006-05-18 US US11/436,457 patent/US7498308B2/en not_active Expired - Fee Related
• 2007
  • 2007-01-11 CY CY20071100040T patent/CY1105917T1/el unknown
• 2009
  • 2009-05-13 CY CY20091100508T patent/CY1109061T1/el unknown

Also Published As

Similar Documents

Legal Events