WO2002085406A9 - Procedes et compositions pour le traitement d'etats lies a la resistance a l'insuline - Google Patents

Procedes et compositions pour le traitement d'etats lies a la resistance a l'insuline

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Publication number
WO2002085406A9
WO2002085406A9 PCT/US2002/013088 US0213088W WO02085406A9 WO 2002085406 A9 WO2002085406 A9 WO 2002085406A9 US 0213088 W US0213088 W US 0213088W WO 02085406 A9 WO02085406 A9 WO 02085406A9
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WIPO (PCT)
Prior art keywords
glp
insulin resistance
week
insulin
glucose
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PCT/US2002/013088
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English (en)
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WO2002085406A1 (fr
Inventor
Jens Juul Holst
Mette Zander Olsen
David R Hathaway
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Restoragen Inc
Jens Juul Holst
Mette Zander Olsen
David R Hathaway
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Application filed by Restoragen Inc, Jens Juul Holst, Mette Zander Olsen, David R Hathaway filed Critical Restoragen Inc
Publication of WO2002085406A1 publication Critical patent/WO2002085406A1/fr
Publication of WO2002085406A9 publication Critical patent/WO2002085406A9/fr

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    • 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

Definitions

  • the present invention relates to endocrinology, physiology and pharmacology. More particularly, it relates to methods and compositions for treating conditions associated with insulin resistance.
  • Insulin resistance is a multi-factorial disease characterized by the requirement of inappropriately high levels of insulin for maintaining glucose homeostasis. It is a relatively common disorder that accompanies obesity, type-2 diabetes, type-2 pre-diabetes, several disorders including essential hypertension, ventricular dysfunction with or without congestive symptoms, post- surgical stress, trauma, polycystic ovary syndrome, burns, gestational diabetes associated with pregnancy, sepsis, myocardial infarction, and stroke, and follows therapy with certain hormones such as glucocorticoids and growth hormone.
  • Insulin resistance is one of the fundamental components of the insulin resistance syndrome, also known as "syndrome X” or "metabolic syndrome". It is characterized by glucose intolerance, hypertriglyceride ia, reduced levels of high-density lipoprotein cholesterol, hyperinsulinemia and central obesity. Other manifestations of the disease include hypertension, albuminuria, increased serum levels of plasminogen activator inhibitor and increased serum uric acid.
  • Insulin resistance is an independent risk factor for the development of atherosclerotic vascular disease, which may cause myocardial infarction, stroke, peripheral vascular disease and death. More recently, the insulin resistance and hyperinsulinemia associated with obesity have been linked to cardiac hypertrophy in young individuals.
  • Metformin and the thiazoladinediones are presently the only drugs available that increase insulin sensitivity. They are effective oral agents for treating the early stages of diabetes, without affecting endogenous secretion of insulin from the pancreas. These drugs may delay or prevent the onset of the later, insulin-dependent phase, as it is widely believed that chronic overproduction of insulin by pancreatic ⁇ -cells secondary to profound insulin resistance leads to depletion of ⁇ -cell mass and subsequent insulin-dependent diabetes.
  • insulin resistance is a risk factor for cardiovascular disease, it often accompanies myocardial infarction and heart failure.
  • insulin resistance develops or is exacerbated by the rise in neurohormones (e.g. catecholamines, glucocorticoids, TNF- ⁇ , etc.) that frequently accompanies these disorders.
  • Metformin and thiazoladinediones are not adequate drugs for treating insulin resistance associated with cardiovascular disease. Metformin is contraindicated in patients with heart disease due to the risk of metabolic acidosis.
  • thiazoladinediones are known to cause fluid retention and can precipitate congestive heart failure in patients with left ventricular dysfunction.
  • Insulin resistance also affects skeletal muscle independently of other diseases. It impairs vasodilatation of skeletal muscle blood vessels, reduces transport capacity of glucose into skeletal muscle cells and alters the ratio of fatty acid oxidation versus glucose oxidation as an energy source for oxidative phosphorylation. These effects reduce exercise capacity and contribute independently to ambulatory limitation in obesity and in various cardiovascular diseases, including heart failure.
  • the available drugs, metformin and the thiazoladinediones have limited use and are contraindicated in patients with heart disease. Accordingly, there is a need for new and better compositions and methods for treating insulin-resistance conditions or disorders.
  • glucagon-like peptide-1 (GLP-1) is capable of reversing insulin resistance.
  • the present invention relates to methods for treating insulin resistance-associated conditions using GLP-1.
  • the insulin resistance-associated condition is selected from the group consisting of type-2 pre- diabetes, atherosclerotic cardiovascular disease (ASCD) , drug-induced insulin resistance, congestive heart failure, diminished exercise capacity of skeletal muscle, and left ventricular dysfunction with cardiac metabolic myopathy or diminished exercise capacity of skeletal muscle, with the proviso that said congestive heart failure is not associated with toxic hypervolemia .
  • the GLP-1 is selected from the group consisting of GLP-1 (7-36) NH 2 , GLP-1 (9- 36)NH 2 , GLP-4(7-37), and exendin-4. [0012] In yet another embodiment, the GLP-1 is administered for a period of time of at least 6, 8, 10, 12 or 14 weeks. In another embodiment, the subject is a human .
  • FIG. 1 Plasma glucose concentrations during 8-h profiles.
  • Fig. 1A represents plasma glucose levels in the GLP-1 and saline group before infusion of GLP-1 and saline respectively was started.
  • Fig. IB represents plasma glucose levels of patients infused with saline at week 0, weekl and week 6.
  • FIG. 2 Plasma glucose levels in GLP-1 treated patients at week 0, weekl and week 6.
  • FIG. 3 Measurement of the insulin levels at weeks 0 and 6 for saline and GLP-1 treated patients. Insulin sensitivity was measured using the hyperinsulinemic euglycemic clamp, before infusion was started (week 0) and after 6 weeks of infusion (week 6) .
  • FIG. 4 Measurement plasma glucose levels during the clamp in Fig. 3 at weeks 0 and 6 for saline and GLP-1 treated patients.
  • FIG. 5 Measurement of insulin sensitivity (mg glucose/kg fat free mass/min) at weeks 0 and 6 for saline and GLP-1 treated patients during the clamp in Fig. 3.
  • FIG. 6 Measurement of ⁇ -cell function (C-peptide levels from the hyperglycemic clamp) .
  • the upper curves represent C-peptide levels in patients receiving saline.
  • the lower curves represent C-peptide levels in GLP-1 treated patients.
  • Incremental area under the curve (AUC) 0-90, first phase responses (incremental AUC 0-10) , second phase responses (incremental AUC 10-45) and maximal secretory capacity (incremental peak levels after glucose plus arginine) all significantly increased. P values are shown in the result section.
  • the small inserted figure represents C-peptide levels from 0-20 minutes .
  • FIG. 7 The effect of dilated cardiomyopathy on norepinephrine, insulin, free fatty acid and glucose levels in a dog model.
  • FIG. 8 The effect of GLP-1 on myocardial free fatty acid/glucose uptake ratio in dilated cardiomyopathy.
  • FIG. 9 The effect of GLP-1 on cardiac function in dilated cardiomyopathy.
  • FIG. 9A represents the effect of GLP-1 on stroke volume.
  • FIG. 9B represents the effect of GLP-1 on left ventricular systolic pressure.
  • FIG. 9C represents the effect of GLP-1 on left ventricular contractility.
  • FIG. 9D represents the effect of GLP-1 on heart rate.
  • GLP-1 GLP-1 molecule
  • Bioly active in this context means having the biological activity of GLP-1 (7-36) amide (GLP-1 (7-36) NH 2 ) , but it is understood that the activity of the variant, analog, mimetic, agonist, or derivative thereof can be either less potent or more potent than native GLP-1 (7-36) amide .
  • insulin resistance refers to a subnormal biological response to a given concentration of insulin.
  • Insulin resistance-associated condition refers to any disease, disorder or condition whereby insulin resistance is a comorbid condition that contributes, exacerbates or interacts synergistically with symptoms, signs, prognosis or clinical outcome.
  • Insulin resistance-associated condition includes but is not limited to type-2 pre-diabetes, ASCD, drug-induced insulin resistance, congestive heart failure which is not associated with toxic hypervolemia, myocardial infarction, stroke, left ventricular dysfunction with cardiac metabolic myopathy and diminished exercise capacity of skeletal muscle.
  • insulin regulating refers to an ability to control the release of insulin into the circulation, in relation to blood glucose and fatty acid levels.
  • the terms "therapeutically or pharmaceutically effective” or “therapeutically or pharmaceutically effective amount” refers to an amount of the compound of this invention required to reduce or lessen the severity of insulin resistance for some period of time. A therapeutically or pharmaceutically effective amount also means the amount required to improve the clinical symptoms .
  • the present invention relates to methods for treating conditions associated with insulin resistance in a subject. The methods include administering to a subject a therapeutically effective amount of GLP-1. The methods of this invention further relate to decreasing insulin resistance using GLP-1.
  • GLP-1 plays a key role in the regulation of plasma glucose homeostasis. It is involved in regulating insulin secretion in relation to blood glucose and/or lipid levels, and inhibiting glucagon release by the pancreas, inhibiting gastric acid secretion and motility, and suppressing appetite and food intake. GLP-1 is a member of the incretin group of secretagogue hormones that are released from the intestinal enteroendocrine cells in response to the ingestion of food. GLP-1 binds to the GLP-1 receptors, some of which are expressed on the ⁇ -cells of the pancreas.
  • GLP-1 Binding of GLP-1 to its receptor triggers an intracellular signaling pathway that results in controlling insulin secretion with concomitant inhibition of glucagon production. This in turn leads to uptake of glucose in muscle and fat cells and the inhibition of hepatic glucose production, all of which lowers blood glucose levels.
  • GLP-1 is also capable of increasing insulin sensitivity.
  • GLP-1 is an intestinally produced peptide hormone. Under physiological conditions, GLP-1 is secreted in response to a meal (Kreymann, B. et al.,
  • GLP-1 secretion is reduced in type-2 diabetic patients (Lugari, R. et al . , Horm . Metab . Res . 32: 424-428 (2000)).
  • Exogenous GLP-1 stimulates insulin secretion, inhibits glucagon secretion and reduces plasma glucose in type-2 diabetic patients (Gutniak, M. et al . , IV. Engl . J. Med. 326: 1316-1322 (1992); Nauck, M.A. et al . , Diabetologia 36: 741-744 (1993) ) .
  • GLP-1 reduces appetite in both obese (Naslund, E. et al . , Int . J. Obes . Rela t . Metab . Disord. 23: 304-311 (1999)) and type-2 diabetic subjects (Toft-Nielsen, M.B. et al . , Diabetes Care 22: 1137-1143 (1999) ) .
  • GLP-1 has been shown to reverse age-dependent decline in ⁇ -cell function in rats (Wang, Y. et al., J. Clin . Invest . 99: 2883-2889 (1997)). It has also recently been shown to stimulate ⁇ -cell proliferation and neogenesis in rats (Perfetti, R.
  • GLP-1 molecule includes the following compounds. Mammalian GLP peptides and glucagon are encoded by the same gene. In the ileum, the precursor is processed into two major classes of GLP peptide hormones, namely GLP-1 and GLP-2.
  • GLP-1 (1-37) has the sequence: His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu- Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1).
  • GLP-1 (1-37) is amidated post- translationally to yield GLP-1 (1-36) NH 2 , which has the sequence : His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr- Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg(NH 2 ) (SEQ ID NO: 2), or is enzymatically processed to yield GLP-1 (7- 37), which has the sequence: His-Ala-Glu-Gly-Thr-Phe-Thr- Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-
  • GLP-1 (7-37) can also be amidated to yield GLP-1 (7- 36) amide, which has the sequence: His-Ala-Glu-Gly-Thr- Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (NH 2 ) (SEQ ID NO: 4).
  • GLP-1 ( 1-36) amide can be processed to GLP-1 (7-36) amide.
  • GLP-1 (7-37) SEQ ID NO: 3
  • GLP-1 (7-36) NH 2 SEQ ID NO: 4
  • DPP IV aminodipeptidase
  • GLP-1 (9-37) which has the sequence: Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val- Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 5), and GLP-1 (9- 36) amide, which has the sequence: Glu-Gly-Thr-Phe-Thr- Ser-Asp-Val-Ser-Ser-Tyr-Leu-
  • GLP-1 molecule includes GLP-1 (1-37), GLP-1 (1-36) NH 2 , GLP-1 (7-
  • GLP-1 (7-36) NH 2 GLP-1 (7-36) amide
  • the present invention includes the use of recombinant human GLP-1 peptides and GLP-1 peptides derived from other species, whether recombinant or synthetic.
  • GLP-1 molecule further denotes biologically active variants, analogs, and derivatives of GLP-1 peptides.
  • "Biologically active,” in this context, means having GLP-1 (7-36) biological activity, but it is understood that the variant, analog, or derivative can be either less or more potent than GLP-1 (7-36) amide, a native, biologically active form of GLP-1. See G ⁇ ke & Byrne, Diabetic Medi cine . 13: 854 (1996).
  • GLP-1 molecules of the present invention also include polynucleotides that express agonists of GLP-1 (i.e., activators of the GLP-1 receptor molecule and its secondary messenger activity found on, inter alia , insulin-producing ⁇ -cells) .
  • GLP-1 mimetics that also are agonists of GLP-1 receptors include, for example, chemical compounds designed or anticipated to activate the GLP-1 receptor.
  • GLP-1 molecules include any molecules, whether they be peptides, peptide mimetics, or other molecules, that bind to or activate a GLP-1 receptor, such as the GLP-1 (7-36) amide receptor, and its second messenger cascade.
  • GLP-1 molecules include species having insulin regulating activity and that are agonists of (i.e., activate), the GLP-1 receptor molecule and its second messenger activity on, inter alia , insulin producing ⁇ -cells.
  • GLP-1 molecules also include peptides that are encoded by polynucleotides that express biologically active GLP-1 variants, as defined herein. Also included in the present invention are GLP-1 molecules that are peptides containing one or more a ino acid substitutions, additions or deletions, compared with GLP-1 (7-36) amide . In one embodiment, the number of substitutions, deletions, or additions is 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 5 amino acids or less or any integer in between these amounts. In one aspect of the invention, the substitutions include one or more conservative substitutions.
  • a “conservative” substitution denotes the replacement of an amino acid residue by another, biologically active similar residue.
  • conservative substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or ethionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • the following table lists illustrative, but non- limiting, conservative amino acid substitutions.
  • GLP-1 peptide variants include the above described peptides which have been chemically derivatized or altered, for example, peptides with non-natural amino acid residues (e.g., taurine, ⁇ - and ⁇ -amino acid residues and D-amino acid residues), C-terminal functional group modifications, such as amides, esters, and C-terminal ketone modifications and N-terminal functional group modifications, such as acylated amines, Schiff bases, or cyclization, as found—for example—in the amino acid pyroglutamic acid.
  • non-natural amino acid residues e.g., taurine, ⁇ - and ⁇ -amino acid residues and D-amino acid residues
  • C-terminal functional group modifications such as amides, esters
  • C-terminal ketone modifications such as acylated amines, Schiff bases, or cyclization, as found—for example—in the amino acid pyroglutamic acid.
  • sequence identity refers to a comparison made between two molecules using standard algorithms well known in the art.
  • the preferred algorithm for calculating sequence identity for the present invention is the Smith-Waterman algorithm, where SEQ ID NO:l [i.e., GLP-1 (1-37)] is used as the reference sequence to define the percentage identity of homologs over its length.
  • SEQ ID NO:l i.e., GLP-1 (1-37)
  • GLP-1 molecules also included in "GLP-1 molecules" of the present invention are six peptides in Gila monster venoms that are homologous to GLP-1. Their sequences are compared to the sequence of GLP-1 in Table 1.
  • a GLP-1 (7-36) amide (SEQ. ID NO: 4)
  • b exendin 3 (SEQ. ID NO:7).
  • c exendin 4 (9-39(NH 2 ) (SEQ. ID NO:8).
  • d exendin 4 (SEQ. ID NO: 9).
  • e helospectin I (SEQ. ID NO: 10).
  • f helospectin II (SEQ. ID NO: 11).
  • g helodermin (SEQ. ID NO:12).
  • h Q 8 , Q 9 helodermin (SEQ. ID NO:13).
  • Peptides (a, b, d, e, f, and g) are homologous at positions 1, 7, 11 and 18. GLP-1 and exendins are further homologous at positions, 4, 5, 6, 8, 9, 15, 22, 23, 25, 26 and 29. In position 2, A, S, and G are structurally similar. In position 3, residues D and E (Asp and Glu) are structurally similar. In positions 22 and 23, F (Phe) and I (He) are structurally similar to Y
  • exendins 3 and 4 are identical in 15 positions and equivalent in 5 additional positions. The only positions where major structural changes are evident are at residues 16, 17, 19, 21, 24, 27, 28 and 30. Exendins also have 9 extra residues at the C-terminus .
  • GLP-1 receptors are cell-surface proteins found, for example, on insulin-producing pancreatic ⁇ - cells; GLP-1 (7-36) receptors and variants thereof have been characterised in the art. Methods of determining whether a chemical or peptide binds to or activates a GLP-1 receptor are known to the skilled artisan.
  • the biological activity of a GLP-1 molecule can be determined by in vi tro and in vivo animal models and human studies, as is well known to the skilled artisan.
  • GLP-1 biological activity can be determined by standard methods, in general, by receptor-binding activity screening procedures, which involve providing appropriate cells that express the GLP-1 receptor on their surface, for example, insulinoma cell lines such as RINmSF cells or INS-1 cells.
  • a GLP-1 receptor may be used.
  • cAMP activity or glucose dependent insulin production can also be measured.
  • a polynucleotide encoding the GLP-1 receptor is employed to transfect cells so that they express the GLP-1 receptor protein.
  • these methods may be employed for screening for a receptor agonist by contacting such cells with compounds to be screened and determining whether such compounds generate a signal (i.e., activate the receptor) .
  • GLP-1 receptor for example, transfected CHO cells
  • Other screening techniques include the use of cells that express the GLP-1 receptor, for example, transfected CHO cells, in a system to measure extracellular pH or ionic changes caused by receptor activation.
  • potential agonists may be contacted with a cell that expresses the GLP-1 receptor and a second messenger response (e.g., signal transduction or ionic or pH changes) , may be measured to determine whether the potential agonist is effective.
  • a second messenger response e.g., signal transduction or ionic or pH changes
  • Polyclonal and monoclonal antibodies can be utilized to detect, purify, and identify GLP-1-like peptides for use in the methods described herein.
  • Antibodies such as ABGA1178 detect intact GLP-1 (1-37) or N-terminally-truncated GLP-1 (7-37) or GLP-1 (7-36) amide .
  • Other antibodies detect the end of the C-terminus of the precursor molecule, a procedure that allows one—by subtraction—to calculate the amount of biologically active, truncated peptide (i.e., GLP-1 (7-37 ) amide) .
  • the GLP-1 molecules of the invention that are peptides that can be made by solid-state chemical peptide synthesis. Such peptides can also be made by conventional recombinant techniques using standard procedures described in, for example, Sambrook & Maniatis.
  • "Recombinant,” as used herein, means that a gene is derived from a recombinant (e.g., microbial or mammalian) expression system that has been genetically modified to contain a polynucleotide encoding a GLP-1 molecule as described herein.
  • the GLP-1 molecule peptides of the present invention may be a naturally purified product, or a product of synthetic chemical procedures, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (for example, by bacteria, yeast, higher plant, insect, or mammalian cells in culture or in vivo) .
  • prokaryotic or eukaryotic hosts for example, by bacteria, yeast, higher plant, insect, or mammalian cells in culture or in vivo
  • the polypeptides of the present invention are generally non-glycosylated, but may be glycosylated.
  • the GLP-1 like peptides can be recovered and purified from recombinant cell cultures by methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (HPLC) can be employed for purification steps.
  • Particularly preferred GLP-1 molecules of the invention are GLP-1 (7-36) amide, GLP-1 (7-37), and exendin- 4.
  • the methods and compositions of this invention may be used to treat conditions associated with insulin resistance.
  • Insulin resistance is associated with several conditions including type-2 pre-diabetes, atherosclerotic cardiovascular disease (ASCD),drug- induced insulin resistance, congestive heart failure which is not associated with toxic hypervolemia, diminished exercise capacity of skeletal muscle, and left ventricular dysfunction with at least one of cardiac metabolic myopathy or reduced exercise capacity of skeletal muscle. Therefore, the present invention provides methods of treating conditions associated with insulin resistance comprising the step of administering
  • Insulin resistance may be due to any one or more events including abnormal prereceptor (e.g., abnormal ligand or competition), receptor (e.g., abnormal structure, affinity of ligand to receptor, or number of receptors), or postreceptor (e.g., abnormal signaling) events.
  • Insulin resistance may be determined by a number of methods known in the art. For example, the euglycemic hyperinsulinemic clamp technique may be used to diagnose insulin resistance (Rao, G., Am . Fam . Physician (2001) 63: 1159-63). This technique involves intravenous administration of an insulin dose while simultaneously maintaining glucose at a pre-set level within the normal range by also administering glucose.
  • the invention provides a method for treating insulin resistance associated with type-2 pre-diabetes comprising the step of administering GLP-1.
  • Insulin resistance has an important role as an independent risk factor for the development of atherosclerotic cardiovascular diseases (ASCD) .
  • ASCD atherosclerotic cardiovascular diseases
  • hyperinsulinemia is an independent risk factor for coronary artery disease.
  • high levels of circulating insulin are associated with hypertension, which may result from higher sympathetic nerve activity.
  • another embodiment of the invention provides a method for treating ASCD comprising the step of administering GLP-1.
  • insulin resistance develops as a consequence of therapy with certain hormones, including but not limited to, glucocorticoids or growth hormone.
  • the invention provides a method for treating drug-induced insulin resistance comprising the step of administering GLP-1.
  • the drug is selected from the group consisting of glucocorticoid and growth hormone.
  • Insulin resistance also develops as an associated disease that contributes to overall morbidity and mortality in other diseases, such as congestive heart failure which is not associated with toxic hypervolemia, left ventricular dysfunction secondary to myocardial injury and diminished skeletal muscle exercise capacity.
  • Neurohormones such as catecholamines, glucagon, cortisol, growth hormone and cytokines such as TNF- ⁇ are all known to induce insulin resistance alone or in combination.
  • these neurohormones initiate insulin resistance in the aforementioned diseases, as associated stress causes many of the neurohormones to be secreted into the blood. In other cases, the neurohormones may simply exaggerate the underlying resistance.
  • Insulin resistance alters the metabolism and function of tissues that use both fatty acids and glucose as substrates for oxidative phosphorylation (e.g., oxygen-dependent ATP synthesis) . This is most evident in cardiac and skeletal muscle. For example, in diseases associated with severely depressed left ventricular dysfunction and reduced peripheral blood flow, insulin resistance develops subsequent to the rise in serum neurohormones. The resistance is tantamount to insulin deficiency.
  • insulin drives the oxidation of glucose via enhanced transport of glucose into muscle and via activation of the enzyme, pyruvate dehydrogenase .
  • Insulin promotes the oxidation of glucose and reduces the oxidation of fatty acids. With insulin resistance the effect is lost and fatty acid oxidation is preferred.
  • Fatty acids are oxidized to produce ATP at lower overall efficiency. That is, more oxygen is required to produce each ATP molecule compared to stoichiometric amounts of glucose .
  • cardiac output is compromised by left ventricular dysfunction (e.g., that may occur subsequent to myocardial ischemia, infarction or cardiomyopathy) the reduced efficiency of fatty acid oxidation may further compromise heart function.
  • left ventricular dysfunction e.g., that may occur subsequent to myocardial ischemia, infarction or cardiomyopathy
  • the reduced efficiency of fatty acid oxidation may further compromise heart function.
  • other changes in cardiac metabolism may amplify the inefficiency problem.
  • the accumulation of long chain acyl-CoA molecules and inhibition of the transport of fatty acids in the mitochondria may uncouple oxidative phosphorylation, causing more oxygen consumption at even lower rates of ATP synthesis.
  • Such patients acquire a new disease, namely, a reversible metabolic myopathy.
  • the invention provides a method for treating insulin resistance associated with a condition selected from the group consisting of congestive heart failure and left ventricular dysfunction with cardiac metabolic myopathy or diminished exercise capacity of skeletal muscle, comprising the step of administering GLP-1; with the proviso that said congestive heart failure is not associated with toxic hypervolemia.
  • Insulin resistance is also associated with impaired skeletal muscle vasodilatation during exercise in the presence or absence of obesity, or left ventricular dysfunction.
  • This impaired skeletal muscle vasodilatation diminishes exercise capacity.
  • glucose uptake and utilization by skeletal muscle are likewise impaired in patients with insulin resistance.
  • the invention provides a method for treating insulin resistance associated with diminished exercise capacity of skeletal muscle, in the presence or absence of obesity, left ventricular dysfunction or congestive heart failure comprising the step of administering GLP-1; with the proviso that said congestive heart failure is not associated with toxic hypervolemia.
  • the insulin resistance associated with diminished exercise capacity is in the presence of obesity, left ventricular dysfunction or congestive heart failure, which is not associated with toxic hypervolemia.
  • the insulin resistance associated with diminished exercise capacity of skeletal muscle is in the absence of obesity, left ventricular dysfunction or congestive heart failure, which is not associated with toxic hypervolemia.
  • the GLP-1 molecules may be formulated into pharmaceutical compositions for administration to subjects, including humans. These pharmaceutical compositions, preferably include an amount of GLP-1 effective to treat insulin resistance and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers useful in these pharmaceutical compositions include, e.g., ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat .
  • compositions of the present invention may be administered parenterally, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions are administered by infusion or subcutaneous injection of a slow-release formulation.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1, 3-butanediol .
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides .
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
  • a suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • Such materials include cocoa butter, beeswax and polyethylene glycols.
  • the pharmaceutical compositions of this invention may also be administered topically. Topical application can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
  • the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
  • the pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • the amount of GLP-1 molecule that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • the compositions can be formulated so that a dosage of between 0.1-1000 pmoles/kg body weight/minute (when administered by infusion) of GLP-1 molecule is administered to a patient receiving these compositions.
  • the dosage is 0.1-10 pmoles /kg body weight/minute when administered by infusion) .
  • the dosage is 0.5 - 2.0 pmoles/kg/min/when administered by intravenous infusion.
  • the composition may be administered as a single dose, multiple doses or over an established period of time in an infusion.
  • the pharmaceutical composition may be administered to a subject for a period of time including, but not limited to, at least 6, 8, 10, 12 or 14 weeks. Other long-term treatments with a pharmaceutical composition of this invention may be suitable.
  • GLP-1 is administered to patients with a confirmed condition associated with insulin resistance.
  • GLP-1 is administered by injection at least once a day or by continuous infusion via pump.
  • GLP-1 is formulated for administration from a subcutaneous depot over a period of days to weeks, oral administration or by intermittent inhalation.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular GLP-1 molecule, the patient's age, body weight, general health, gender, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within ordinary skill in the art.
  • the amount of GLP-1 molecule will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.
  • the amounts of GLP-1 molecules can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • Recombinant GLP-1 (7-36) amide (BioNebraska, Lincoln, Iowa) was supplied as a 1 mg/ l liquid sterile formulation, with a peptide purity of >99% as determined by reversed-phase HPLC The infusion rate was 4.8 pmol/kg/min.
  • the portable insulin pump was to be used continuously (day and night) for the next 6 weeks.
  • Patients receiving GLP-1 were instructed about filling up their pumps every 24 hours.
  • Patients receiving saline had their pumps filled at the hospital once a week.
  • the patients underwent a hyperglycemic clamp, measurement of gastric emptying and 8-h profiles were obtained. After 6 weeks of infusion (week 6) , all procedures from week 0 were repeated.
  • GLP-1 was measured twice daily on examination days and once weekly between week 1 and week 6 in order to monitor pump function and patient compliance.
  • HbA ⁇ c was measured at week 0 and 6, fructosamine at week 0, 3 and 6. Between week 1 and 6, the patients were monitored for control of blood glucose, body weight, blood pressure and pulse.
  • Body composition and weight was estimated by a DXA scanner, Norland XR 36 (Norland Instruments, Fort Atkinson, WI, USA). See Gotfredsen, A. et al . J. Appl . Physiol . 82: 1200-1209 (1997), incorporated herein by reference .
  • Insulin was administered as a primed infusion at a rate of 40 mU/m 2 /minute in order to raise the plasma insulin level by 100 ⁇ U/ml. See DeFronzo, R.A. et al.
  • Plasma glucose, insulin, C-peptide, glucagon and free fatty acid (FFA) levels were determined every half hour.
  • Plasma glucose concentrations were analyzed using a Beckman Analyzer (Beckman Instrument, Fullerton, CA) .
  • the glucagon assay (Orskov, C et al . J. Clin . Invest . 87: 415-423 (1991)) is directed against the COOH- terminus of the glucagon molecule (antibody code no. 4305) .
  • Total GLP-1 was measured by radioimmunoassay employing antiserum code no. 89390 which is highly specific for the COOH-terminus of GLP-1 and therefore measures the sum of GLP-1 (7-36) amide and its metabolite GLP-1 (9-36) amide (Orskov, C . , et al.
  • glucagon and GLP-1 analysis plasma was extracted with ethanol (final concentration 70%vol/vol) before analysis. Detection limits and intra-assay coefficients of the assays used were 1 pmol/1 and ⁇ 6% for glucagon, 1 pmol/1 and ⁇ 6% for GLP-1 (antibody code no. 89390) . Insulin and C-peptide concentrations were measured using commercial ELISA kits (code No K6219 and K6218 respectively, Dako ®, Copenhagen, Denmark) . Detection limits of the assays were 3 pmol/1 for insulin and 17 pmol/1 for C-peptide.
  • Intra- and interassay coefficients of variation were 4-10 % at 39-1240 pmol/1 for insulin and 3-6 % at 380 -2700 pmol/1 for C-peptide.
  • the crossreactivity with intact and split proinsulins in the C-peptide assay was 63-87 %.
  • HbA ⁇ c was measured by ion-exchange HPLC (at Steno Diabetes Hospital, Gentofte, Denmark) with an interassay coefficient of variation of 0.15 % in the range of 4.7-11.3 % (normal range: 4.1-6.4 %) .
  • FFAs were measured using an enzymatic colorimetric assay (Wako, WA, USA) .
  • Plasma levels of GLP-1 in patients who had been administered saline remained unaltered at 10.8+2.4 (week 0) versus 10.5 ⁇ 2.3 (week 1) versus 9.4 ⁇ 2.1 (week 6) pmol/1, NS.
  • GLP-1 increased from 19.0 ⁇ 9.7 (week 0) to 196.8+27.9 (week 1) and 253.7 ⁇ 46.1 (week 6), P ⁇ 0.0001 in patients treated with GLP-1.
  • ⁇ values (changes in patients receiving GLP-1 versus changes in controls): P ⁇ 0.0001.
  • 8-h plasma glucose profiles are provided in Figure 1.
  • Plasma glucose levels remained unchanged over the 6 weeks ( Figure IB) .
  • fasting and 8-h mean plasma glucose levels decreased by 3.4 and 4.9 mmol/1 (week 1), respectively, and by 4.3 and 5.5 mmol/1 (week 6), respectively, P ⁇ 0.001 ⁇ values: P ⁇ 0.0001 ( Figure 2).
  • ⁇ values: P 0.001.
  • the decline in HbA lc would probably have been more pronounced if the GLP-1 treatment had lasted longer than 6 weeks, since it takes more than six weeks to reach new steady state levels of HbA ⁇ c .
  • FIG. 6 shows C-peptide levels during the hyperglycemic clamp.
  • incremental AUC C-peptide levels remained unaltered.
  • Incremental maximal secretory capacity peak levels after arginine infusion
  • ⁇ 0.0001 ⁇ values: P ⁇ 0.0001.
  • Dilated cardiomyopathy was induced in conscious dogs by rapid ventricular pacing at 210 beats per minute over 28 days. As shown in Figure 7, the pacing was associated with a significant rise in serum glucose (9012 to 115+4 mg%), insulin (27+2 to 7518 pmol/1), free fatty acids 269+43 to 702+50 ⁇ mol/1) and norepinephrine (83+8 to 320+48 pg/ml) . All increases were statistically significant. The combination of elevated glucose in the presence of increased levels of insulin is pathognomonic of insulin resistance.
  • Figure 8 demonstrates the changes in uptake of free fatty acids versus glucose into heart muscle that occur with cardiomyopathy measured at 28 days of pacing and following 48 hours of treatment with placebo or GLP-1 (5 ng/kg/min) .
  • the ratio of uptake of free fatty acids (FFA) to glucose was nearly 1.0, indicating approximately equal uptake.
  • the ratio is approximately 1.5, indicating that FFA are preferred over glucose.
  • the ratio is reversed (0.5), indicating that GLP-1 preferentially enhances the uptake of glucose over FFA in the cardiomyopathy heart.
  • Figure 9 demonstrates the physiological consequences of GLP1 in pacing-induced cardiomyopathy.
  • GLP-1 increases stroke volume (p ⁇ 0.001), left ventricular (LV) systolic pressure (p ⁇ 0.001) and LV dP/dT (p ⁇ 0.001) and decreases heart rate (p ⁇ 0.005).
  • LV left ventricular
  • LV dP/dT LV dP/dT

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Abstract

L'invention concerne des procédés et des compositions destinés au traitement d'états associés à l'insuline, qui consistent à administrer du peptide-1 de type glucagon (GLP-1) à des patients qui en souffrent.
PCT/US2002/013088 2001-04-24 2002-04-24 Procedes et compositions pour le traitement d'etats lies a la resistance a l'insuline WO2002085406A1 (fr)

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US7105489B2 (en) * 2002-01-22 2006-09-12 Amylin Pharmaceuticals, Inc. Methods and compositions for treating polycystic ovary syndrome
US7790681B2 (en) 2002-12-17 2010-09-07 Amylin Pharmaceuticals, Inc. Treatment of cardiac arrhythmias with GLP-1 receptor ligands
AU2003297356A1 (en) * 2002-12-17 2004-07-14 Amylin Pharmaceuticals, Inc. Prevention and treatment of cardiac arrhythmias
JP4565193B2 (ja) 2003-04-23 2010-10-20 バレリタス， インコーポレイテッド 長い持続時間の医薬投与のための液圧作動式ポンプ
WO2006014425A1 (fr) 2004-07-02 2006-02-09 Biovalve Technologies, Inc. Procedes et dispositifs pour l'administration du glp-1 et leurs utilisations
WO2007115039A2 (fr) 2006-03-30 2007-10-11 Valeritas, Llc dispositif d'acheminement de fluide à cartouches multiples
DK2344519T3 (en) 2008-11-07 2017-01-23 Massachusetts Gen Hospital C-TERMINAL FRAGMENTS OF GLUCAGON SIMILAR PEPTID-1 (GLP-1)
WO2012061466A2 (fr) 2010-11-02 2012-05-10 The General Hospital Corporation Méthodes de traitement d'une maladie de stéatose
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