EP1461068A1 - Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens - Google Patents

Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens

Info

Publication number
EP1461068A1
EP1461068A1 EP02803945A EP02803945A EP1461068A1 EP 1461068 A1 EP1461068 A1 EP 1461068A1 EP 02803945 A EP02803945 A EP 02803945A EP 02803945 A EP02803945 A EP 02803945A EP 1461068 A1 EP1461068 A1 EP 1461068A1
Authority
EP
European Patent Office
Prior art keywords
cgrp
receptor
agonist
amylin
high affinity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02803945A
Other languages
German (de)
English (en)
Other versions
EP1461068A4 (fr
Inventor
Garth James Smith Cooper
Kerry Martin Loomes
Rachel Nancy Watson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PHILERA NEW ZEALAND Ltd
Original Assignee
Protemix Corp Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Protemix Corp Ltd filed Critical Protemix Corp Ltd
Publication of EP1461068A1 publication Critical patent/EP1461068A1/fr
Publication of EP1461068A4 publication Critical patent/EP1461068A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • A61K38/225—Calcitonin gene related peptide
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • 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
    • A61P9/12—Antihypertensives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • This invention provides methods and compositions for modulating lipid levels in a cell or tissue, and for identifying agents useful for modulating lipid levels.
  • the invention finds use in the fields of biology and medicine.
  • a high-fat diet influences insulin-stimulated posttransport muscle glucose metabolism in rats. Metabolism. 1997 Sep;46(9): 1101-6; Dobbins et al.. Prolonged inhibition of muscle carnitine palmitoyltransferase-1 promotes intramyocellular lipid accumulation and insulin resistance in rats. Diabetes. 2001 Jan;50(l):123-30; Kraegen et al. Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats. Diabetes. 1991 Nov;40(l l):1397-403; Unger et al.. Lipotoxic diseases of nonadipose tissues in obesity. Int J Obes Relat Metab Disord. 2000 Nov;24 Suppl 4:S28-32; Kelley et al.
  • Insulin resistance is a primary pathogenic process for several major metabolic diseases of humans with common underlying pathogenic mechanisms, referred to as the "metabolic syndrome" or "Syndrome X complex.”
  • Diseases commonly included within Syndrome X complex include type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis, and thrombosis (see, e.g., Zierath et al., 2000, Diabetologia 43:821-35; Bergman et al., 2001, J Investig Med. 49:119-26; Olefsky et al., 1995, Am J Clin Nutr.
  • the invention provides a method of stimulating lipolysis in a tissue of a mammal (e.g., skeletal muscle or liver) by contacting the tissue with an agonist of the high affinity CGRP receptor in an amount of agonist effective to preferentially stimulate activity of a high affinity CGRP receptor compared to the metabolic amylin (e.g., stimulate activity of a high affinity CGRP receptor without substantially stimulating activity of a metabolic amylin receptor).
  • the agonist is CGRP-1.
  • the tissue is contacted with CGRP-1 at between about 10 "15 M and about 10 "10 M.
  • the amount is at less than 300 pM.
  • the tissue is isolated.
  • the method includes the step of detecting stimulation of lipolysis in the tissue.
  • the invention provides a method of stimulating lipolysis in a mammalian cell, such as a skeletal muscle cell, by contacting the cell with a CGRP polypeptide or biologically functional variant thereof.
  • the CGRP polypeptide has the sequence of a naturally occurring CGRP polypeptide, such as a human CGRP.
  • the CGRP polypeptide is a human CGRP-1 polypeptide, or biologically functional variant thereof.
  • the polypeptide or variant preferentially stimulates the high affinity CGRP receptor.
  • the invention provides a method of stimulating lipolysis in skeletal muscle or liver of a mammal by contacting the skeletal muscle or liver with CGRP-1 or a metabolic receptor stimulating variant thereof.
  • the method may further comprise monitoring a change in the level of lipolysis in a tissue of the mammal (e.g., by measuring the amount of free fatty acids in muscle, liver, blood or other tissue of the mammal).
  • the invention provides a therapeutic regimen including (i) administering a CGRP-1 polypeptide, a biologically function variant thereof, or a metabolic receptor stimulating variant thereof to a mammal suffering from or susceptible to a condition characterized by accumulation of lipid in skeletal muscle, and (ii) monitoring lipolysis in the mammal.
  • the invention provides a method of reducing skeletal muscle lipid levels in a mammal in need of such treatment by administering an agonist of the high affinity CGRP receptor under conditions in which the metabolic amylin receptor is not stimulated by the agonist or the high affinity receptor is preferentially stimulated, wherein the mammal has a condition or disease characterized by lipid accumulation in a tissue.
  • the mammal is insulin resistant.
  • the method further includes detecting a change in the amount of free fatty acids in a tissue (e.g., skeletal muscle, liver or blood) of the mammal.
  • the method further includes monitoring the course of the condition or disease.
  • the administration of the agonist does not result in an increase in blood glucose levels in the mammal.
  • the invention provides a method of reducing lipid levels in a tissue (e.g., skeletal muscle or liver) by contacting the tissue with a CGRP polypeptide or biologically functional variant thereof.
  • a tissue e.g., skeletal muscle or liver
  • the CGRP polypeptide or variant is administered to a mammal in an amount effective to reduce tissue lipid levels.
  • the mammal is insulin resistant and the polypeptide or variant is administered in an amount effective to reduce insulin resistance in the mammal.
  • the polypeptide or variant preferentially stimulates the high affinity CGRP receptor.
  • the invention provides use of a CGRP polypeptide, such as CGRP-1, or a biologically functional variant thereof in the preparation of a medicament for reducing lipid levels in skeletal muscle of a mammal or in the preparation of a medicament for treatment of insulin resistance.
  • the invention provides a method of stimulating lipolysis in a cell that expresses a high affinity CGRP receptor, by contacting the cell with an agonist of the high affinity CGRP receptor.
  • the agonist preferentially activates the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • the invention provides a method of stimulating lipolysis in a mammal by administering to the mammal an agonist of the high affinity CGRP receptor.
  • the agonist preferentially activates the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • the agonist is a polypeptide, such as a CGRP polypeptide or biologically functional variant thereof.
  • the method includes measuring the amount of free fatty acids in a tissue (skeletal muscle, liver, serum, plasma or blood) of the mammal or monitoring the effect of the administration of CGRP to the mammal on insulin resistance in the mammal.
  • the agonist is administered in an amount effective to stimulate lipolysis without stimulating vasodilatation.
  • the invention provides a method of stimulating lipolysis in a tissue (e.g., skeletal muscle or liver) in a mammal in need of such treatment, by administering an amount of an agonist of the high affinity CGRP receptor sufficient to preferentially stimulate the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • the mammal to which an agonist is administered has a disease or condition characterized by lipid accumulation in skeletal muscle (e.g., insulin resistance, Syndrome X, type-2 diabetes) or in liver (e.g., hepatic steatosis or "fatty liver").
  • the agonist is administered with a pharmaceutically acceptable carrier.
  • the invention also provides the use of an agonist of the high affinity CGRP receptor (such as a CGRP-1 polypeptide) for the preparation of a medicament for treatment of a condition or disease (e.g., insulin resistance or Syndrome X) characterized by lipid accumulation in a tissue (e.g., skeletal muscle or liver) of a mammal.
  • a condition or disease e.g., insulin resistance or Syndrome X
  • the invention provides the use of CGRP-1 in the preparation of a medicament for treating a mammal suffering from or susceptible to a condition characterized by accumulation of lipid in a tissue (e.g., skeletal muscle or liver), where the medicament when administered to the mammal results in a level of agonist in blood that is less than 300 pM or when administered to the mammal results in a level of agonist in blood that is less between about 10 "15 M and about 10 "10 M.
  • a tissue e.g., skeletal muscle or liver
  • the invention provides the use of CGRP-1 in the preparation of a medicament for treating a mammal suffering from or susceptible to a condition characterized by accumulation of lipid in a tissue (e.g., skeletal muscle or liver), where the medicament when administered to the mammal results in a level of agonist in blood mat is between about 10 "15 M and about 10 0 M.
  • a tissue e.g., skeletal muscle or liver
  • the condition is diabetes, insulin resistance, or Syndrome X.
  • the invention also provides methods of determining whether an agent useful for stimulating lipolysis in a mammal by determining whether the agent is an agonist of a CGRP receptor, such as the high affinity CGRP receptor. In an embodiment, the method further includes determining that the agent preferentially stimulates the high affinity CGRP receptor compared to the metabolic amylin receptor. In an embodiment, the method further includes comparing the EC 50 of the agent for effecting a response mediated by the metabolic amylin receptor (e.g., an effect on carbohydrate metabolism) and the EC 50 of the agent for effecting a response mediated by the high affinity CGRP receptor (an increase in free fatty acids in skeletal muscle tissue or a skeletal muscle cell). In an embodiment, the EC 50 values are determined in vitro using isolated skeletal muscle. In an embodiment, the effect carbohydrate metabolism is a change in tissue or serum glucose and/or lactate levels and/or tissue glycogen levels.
  • the invention also provides a method of determining whether a compound is useful as a therapeutic agent by determining whether an agent stimulates lipolysis in a mammalian tissue that expresses the high affinity CGRP receptor and the metabolic amylin receptor; and determining whether a lipolysis stimulating agent preferentially stimulates the high affinity CGRP receptor compared to the metabolic amylin receptor, where an agent that preferentially stimulates the high affinity CGRP receptor is determined to be useful as a therapeutic agent.
  • This and other methods of the invention can be used to screen for compounds useful as therapeutic drug.
  • drug screening includes carrying out the aforementioned method with at least 25 different agents (e.g., simultaneously or sequentially).
  • the invention provides a screening method that involves (i) identifying a plurality of agents that bind the high affinity CGRP receptor; and, (ii) selecting an agent from (i) that preferentially stimulates the high affmity CGRP receptor compared to the metabolic amylin receptor.
  • the method involves (i) identifying a plurality of agents that bind the high affrnity CGRP receptor; and, (ii) selecting an agent from (i) that has an EC 50 for effecting a response mediated by the metabolic amylin receptor which is higher than the EC 50 of the agent for effecting a response mediated by the high affinity CGRP receptor.
  • the EC 50 values are determined in vitro using isolated skeletal muscle (e.g., from rat).
  • the EC 50 for effecting a response mediated by the metabolic amylin receptor is at least 10-fold higher than the EC 50 of the agent for effecting a response mediated by the high affinity CGRP receptor.
  • the response mediated by the high affinity receptor can be an increase in free fatty acids in skeletal muscle tissue or a skeletal muscle cell and the response mediated by the metabolic amylin receptor can be an effect on carbohydrate metabolism.
  • the invention provides a method of determining whether an agent useful for stimulating lipolysis in a mammal by (i) identifying a plurality of agents that stimulate lipolysis in a tissue expressing the high affinity CGRP receptor; and, (ii) selecting an agent from (i) that preferentially stimulates the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • the tissue is skeletal muscle (e.g., from rat).
  • the library screened has more than about 100 different test agents.
  • the invention also provides a method for determining whether an agent is useful for stimulating lipolysis in a mammal comprising comparing the lipolytic activity of the agent with the lipolytic activity of CGRP.
  • the invention also provides a method of treating insulin resistance in a mammal by administering a CGRP receptor agonist in an amount effective to activate a high affinity CGRP receptor in a tissue of the mammal without activating the metabolic amylin receptor, where the agonist is an agent identified using a screening method described above.
  • the treatment method ncludes the additional step of measuring the amount of free fatty acids in a tissue of the mammal or of monitoring insulin resistance in the mammal.
  • the invention provides a method of determining a dose or formulation of an agonist of the high affinity CGRP receptor that stimulates lipolysis in a tissue (e.g., skeletal muscle or liver) of a mammal while minimizing undesired side-effects in the mammal (e.g., an increase in blood glucose levels in the mammal or stimulation of vasodilatation in the mammal) by (i) conducting dose- response assays by (a) administering a plurality of different doses or formulations of the CGRP receptor agonist to test mammals; and (b) measuring the effect of each dose or formulation on lipolysis in a tissue of the test mammal and measuring the effect of each dose on the side-effect, thereby creating dose-response data for lipolysis and the side-effect; and, (ii) determining from the dose-response data a dose of the CGRP receptor agonist formulation that increases lipolysis but does not elicit the side-
  • the invention also provides a composition containing a therapeutically effective amount of an agent that preferentially stimulates the high affinity CGRP receptor compared the metabolic amylin receptor, and pharmaceutically acceptable excipient.
  • the composition when administered to a mammal, stimulates the high affinity CGRP receptor in the mammal without stimulating the metabolic amylin receptor.
  • the composition stimulates lipolysis while minimally stimulating vasodilatation.
  • the invention provides a pharmaceutical composition in unit dosage form for administration to a mammal, said unit dosage including an agonist of the high affinity CGRP receptor in an amount sufficient result in a level of agonist in blood sufficient to preferentially stimulate activity of a high affinity CGRP receptor compared to the metabolic amylin (e.g., stimulate activity of a high affinity CGRP receptor without substantially stimulating activity of a metabolic amylin receptor.
  • the agonist is CGRP-1) and a pharmaceutically acceptable excipient.
  • the agonist is CGRP-1 or a biologically functional variant thereof.
  • the agonist is CGRP-1 and the level of agonist in blood is less than 300 pM.
  • the agonist is CGRP-1 and the level of agonist in blood is between about 10 "15 M and about 10 "10 M.
  • the invention further provides the use of an agonist of the high affinity CGRP-1 receptor in the preparation of a medicament for treating a mammal suffering from or susceptible to a condition characterized by accumulation of lipid in a tissue (e.g., skeletal muscle or liver) , wherein the medicament when administered to the mammal results in a level of agonist in the mammal that is sufficient to preferentially stimulate activity of a high affinity CGRP receptor compared to the metabolic amylin (e.g., stimulate activity of a high affinity CGRP receptor without substantially stimulating activity of a metabolic amylin receptor).
  • the agonist is CGRP-1).
  • the invention provides a method of inhibiting lipolysis in a mammal by administering to the mammal an antagonist of the metabolic amylin receptor and/or high affinity CGRP metabolic amylin receptor (e.g., ' amylin or ' CGRP) in an amount effective to inhibit lipolysis.
  • an antagonist of the metabolic amylin receptor and/or high affinity CGRP metabolic amylin receptor e.g., ' amylin or ' CGRP
  • the invention provides a method of stimulating lipolysis in a tissue of a mammal (e.g., skeletal muscle) by contacting the tissue with an agonist of the metabolic amylin receptor (e.g., CGRP).
  • Figure 1 shows amino acid sequences of peptides used in the study.
  • Peptides used are rat amylin, rat CGRP-1 (CGRPf), rat amylin -(8-37) and human CGRP -(8-37). All peptides have an intra-molecular disulfide bond between the 2 nd and 7 th Cys residues.
  • the "NH 2 at the carboxy terminus represents amidation of the hormones.
  • Figure 2A shows effects of amylin, CGRP, norepinephrine and insulin on muscle free fatty acid content.
  • Figure 3B shows effects of amylin- (8-37), CGRP- (8-37) and insulin on the ability of CGRP-1 to increase intramuscular free fatty acids. Soleus muscles were incubated for lh in KHB buffer (control), 100 nM CGRP, 100 nM CGRP with 10 ⁇ M human CGRP-(8-37) (C/C) or amylin - (8-37) (C/A), 10 ⁇ M human CGRP- (8-37) (C-8-37) or 100 nM CGRP + 23.7 nM insulin (C + I). Values are means ⁇ S.E.M.
  • Figure 3C shows effects of amylin-(8-37) and CGRP-(8-37) on the ability of CGRP-1 to increase intramuscular free fatty acids. Soleus muscles were incubated for lh in KHB (control), 1 pM CGRP, lpM CGRP + 100 pM CGRP-(8-
  • Figures 4A and 4B show dose dependent effects of CGRP-1 to stimulate free fatty acid content in incubated rat soleus muscle.
  • Figure 4C shows the effects of CGRP-2 to stimulate free fatty acid content in incubated rat soleus muscle.
  • Figure 5A shows dose dependent effects of amylin to stimulate free fatty acid content in incubated rat soleus muscle. Soleus muscles were incubated for lh in KHB (control) or a range of amylin concentrations. Values are means + S.E.M.
  • Figures 6A, 6B and 6C show effects of amylin, CGRP and norepinephrine on the soleus muscle triglyceride content of rats fed high fat diets. Rats were fed a diet consisting 40% lard (6A), corn oil (6B) or olive oil (6C). Soleus muscles were incubated for lh in KHB (control), 100 nM amylin or CGRP-1 or norepinephrine
  • Figure 8 shows total and non-specific binding of [ 3 H]rat amylin or [ H] salmon calcitonin (sCT) to cultured L6 myoblasts that were transiently transfected with vectors containing inserts as described.
  • Myoblasts were transfected as follows: A, vector alone; B, vector containing murine ramp 1; c, vector containing the insert-negative isoform of the rat calcitonin receptor 1, Cl ins- ; and D, co- transfection with vectors containing murine ramp 1 and murine Cl ins- , respectively.
  • Binding was performed with concentrations of 10 nM of each radioligand in the absence (total (T)) or presence (non-specific (NS)) of 1 ⁇ M concentrations of corresponding unlabelled peptides, as shown. Specific binding may be obtained by subtraction of non-specific from the corresponding total binding in each case. Each bar represents the mean ( ⁇ S.E.M.) of binding derived from three independent myoblast transfections from an experiment repeated at least twice. *, p ⁇ 0.05; ***, p ⁇ 0.001; significance of differences between total and corresponding non-specific binding was determined using unpaired Student's t-tests.
  • Figure 9 shows displacement of bound [ H]rat amylin from L6 myoblasts co-transfected with vectors containing murine ramp 1 and the insert-negative rat calcitonin receptor isoform 1 (Clizie s -).
  • Myoblasts co-transfected with murine ramp 1 and Cl ins - were incubated with [ 3 H] amylin (20 nM) in the presence of indicated concentrations of unlabelled ligands, for 3 h at 21 °C. Cells were then washed and bound radioactivity determined by liquid scintillation counting.
  • Figure 10 shows effects of the truncated peptide antagonists ' rat CGRP-
  • Figure 11 shows specific binding, expressed as a percentage of total binding, of A, [ 3 H]rat CGRP-1, CGRP B,; [ 3 H]rat amylin, amylin; and C, [ 3 H]-sCT, to Cos-7 cells transiently transfected with vector constructs as indicated.
  • Specific binding was derived from differences between total binding of the respective radioligands (20 nM in each case) in the absence or presence of corresponding unlabeled ligands (1 ⁇ M). Each point represent the mean ⁇ SEM of three independent transfections from an experiment performed at least twice. **, p ⁇ 0.01 for binding compared with that to cells transfected with vector alone.
  • Figure 12 shows Northern blots illustrating expression of RNA corresponding to murine receptor activity modifying protein 1, ramp 1, or murine ramp 3, in Cos-7 cells transiently transfected with stated vector constructs.
  • (Upper panel) Northern blots of RNA extracted from COS-7 cells that had been transfected with vector alone, vector; or vector containing inserts corresponding to murine ramp 1 or ramp 3, then probed with labeled cDNAs corresponding to ramp 1 (left panel) or ramp 3 (right panel).
  • Figure 13 shows concentration-dependent effects of CGRP and amylin on cAMP in rat skeletal muscle. Soleus muscle strips were incubated in vitro with rat CGRP-1 (13A), or rat amylin (13B, 13C), at the concentrations shown. Incubations were performed in the absence or presence of human insulin (23.7 nM). Statistical significance was tested by one-way ANOVA, followed by post-hoc analysis using Dunnett's Multiple Comparisons Test, A, * p ⁇ 0.05, **p ⁇ 0.01, compared to control; and B, * p ⁇ 0.05, **p ⁇ 0.01 compared to control.
  • FIG. 14 shows effects of peptide antagonists on amylin-mediated suppression of insulin-stimulated glucose transport in rat soleus muscle in vitro. Rat soleus muscle was isolated, stripped and incubated with amylin (10 nM) and insulin (23.7 nM), as well as with the indicated concentrations of A, 8 ' 37 rat amylin or B, i
  • Figure 15 shows the effect of CGRP and related peptides on basal and insulin-stimulated in vitro metabolism in rat skeletal muscle.
  • Dose-response curves were derived for: (A, B), total glycogen content; and (c, D), rates of incorporation of D[ 14 C(U)]glucose into glycogen, in isolated rat soleus muscle strips incubated in the presence of indicated concentrations of rat amylin, amylin (O); rat CGRP-1, CGRP ( ⁇ ); or salmon calcitonin, sCT (E). Incubations were performed in the absence (A, c), or presence (B, D), of maximally effective human insulin (23.7 nM).
  • FIG. 16 shows insulin dose-repsonse curves in soleus muscle from rats fed a high fat diet. Insulin dose response curves were measured in soleus muscle from rats fed a normal diet ( ⁇ ) or a high fat diet ( ⁇ ) for 51 days. Insulin responses were measured through incorporation of D[ 14 C(U)] glucose into muscle glycogen following incubation for 2 h with various concentrations of insulin.
  • Figures 17A and 17B show the effect of CGRP-1 on muscle and triglyceride content in high- fat fed animals.
  • Figure 18 shows the effect of receptor antagonists of CGRP on CGRP-1 effects on muscle lipid in high fat-fed rats
  • the statistical significance of the results shown was analyzed by one way ANOVA and post-hoc analysis using Tukey's test. *** p ⁇ 0.001, * P ⁇ 0.05 compared to basal; *** P ⁇ 0.001 compared to 100 nM CGRP; !!! P ⁇ 0.001 compared to 1 pM CGRP.
  • Figure 19 shows the effects of CGRP-1 on cAMP content in soleus muscle from normal fed and high fat-fed animals.
  • Figure 20 shows a drop in mean arterial pressure in Wistar rats infused with rat CGRP 1 (100 pmol kg/min) or saline for lh. Mean arterial pressure was continuously measured using a solid-state blood pressure transducer, and monitored using a PowerLab/16s data acquisition module. Calibrated signals were displayed on screen and saved to disc as 2 s averages of each variable. * P ⁇ 0.05.
  • Figure 21 shows the effect on blood glucose levels of infusion with CGRP-1 or an antagonist of CGRP-1 activity. Blood glucose levels were determined at 5-minute intervals using an Advantage meter and tail blood samples from animals infused for 90 minutes as indicated.
  • lipolysis refers to enzymatic hydrolysis of lipids or fat storage compounds such as triglycerides, causing the release of free fatty acids.
  • free fatty acids (FFA) and “non-esterified fatty acids (NEFA)” are used interchangeably herein.
  • terapéuticaally effective amount refers to a predetermined amount of an agent calculated to elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, physician or other clinician, e.g., an amount sufficient to stimulate lipolysis or reduce lipid levels and/or ameliorate a disease state or symptoms, or otherwise prevent, hinder, retard or reverse the progression of a disease or any other undesirable symptoms to achieve a desired therapeutic effect.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” refers to a carrier that does not cause an adverse physical reaction upon administration and one in which a therapeutic agent is sufficiently soluble to deliver a tlierapeutically effective amount.
  • excipients include buffered water, physiological saline, PBS, dextrose solution, Hank's solution and inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate.
  • mammal has its usual meaning and includes primates (e.g., humans and nonhuman primates), experimental animals (e.g., rodents such as mice and rats), farm animals (such as cows, hogs, sheep, and horses), and domestic animals (such as dogs and cats).
  • primates e.g., humans and nonhuman primates
  • experimental animals e.g., rodents such as mice and rats
  • farm animals such as cows, hogs, sheep, and horses
  • domestic animals such as dogs and cats.
  • treatment means (i) preventing the condition or disease, that is, avoiding any clinical symptoms of the disease; (ii) inhibiting the condition or disease, that is, arresting the development or progression of clinical symptoms; and/or (iii) relieving the condition or disease, that is, causing the regression of clinical symptoms.
  • EC 50 has its normal meaning in the art and refers to a concentration of a compound that results in 50% of maximum enhancement of a specified biological effect, e.g., the concentration at which a biological effect mediated by binding of a ligand (e.g., CGRP) to a receptor (e.g., the high affinity CGRP receptor) is at one-half of its maximum value.
  • a ligand e.g., CGRP
  • a receptor e.g., the high affinity CGRP receptor
  • the EC 50 for stimulation of skeletal muscle lipolysis by CGRP and the high affinity CGRP receptor is the concentration of CGRP that results in 50% of maximum enhancement of skeletal muscle lipolysis over a baseline level.
  • the ligand may or may not be naturally occurring.
  • a receptor "agonist” is an agent (compound or composition) capable of promoting at least one of the biological responses normally associated with binding of a natural ligand of the receptor to the receptor.
  • an agonist of the high affinity CGRP receptor present in skeletal muscle cells is an agent (e.g., CGRP) that interacts with (e.g., binds) the high affinity receptor and increases lipolysis in the muscle cells in a dose-dependent manner.
  • a binding interaction between an agonist and a CGRP receptor can be determined by the ability of the agonist to compete with CGRP (e.g., radiolabeled CGRP) for binding to the receptor and/or using other competition assays.
  • a receptor is "stimulated” or, equivalently, is “activated” or, equivalently, is “agonized” by a compound or agent when binding of the compound or agent to the receptor (e.g., to the ligand binding site) results in a change in the metabolic state of the cell expressing the receptor.
  • the ligand e.g., a natural ligand or other agonist
  • the receptor transduces a signal to the cell interior.
  • Grammatical equivalents e.g., activation, stimulation, etc.
  • conservative substitution refers to a change in the amino acid composition of the protein in which residues are replaced with sfructurally similar substitutes that do not substantially alter the protein's activity.
  • conservatively substituted variants refers to amino acid substitutions of those amino acids that are not critical for protein activity, or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter activity.
  • Conservative substitution Tables providing functionally similar amino acids are well known in the art.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see also, Creighton (1984) Proteins, W.H. Freeman and Company).
  • substitutions are not the only possible conservative substitutions. For example, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be "conservatively substituted variants.”
  • peptide refers to a polypeptide fewer than 50 residues in length.
  • protein and “polypeptide” encompass both “peptides” and longer amino acid polymers.
  • the term "contacting,” has its normal meaning of bringing in proximity.
  • Methods of contacting an compound and a receptor expressed by a cell or tissue include, without limitation, incubating the cell or tissue ex vivo (e.g., in vitro) together with the compound under conditions under which the compound can bind the receptor, and administration of the compound to an animal such that the compound is delivered to the cell or tissue by circulatory or other system of the animal.
  • the agent administered is converted to the agonist by metabolic activity of the animal or tissue (e.g., a metabolite of the administered agent is an agonist).
  • the terms “peptide mimetics” or “peptidomimetics” (Fauchere, 1986, Adv. Drug Res.
  • peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as naturally- occurring CGRP polypeptide, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: ⁇ CH 2 NH ⁇ , ⁇ CH 2 S ⁇ , - -CH 2 -CH 2 --, ⁇ CH.dbd.CH-(cis and trans), -COCH 2 --, ⁇ CH(OH)CH 2 --, and ⁇ CH 2 SO--, by methods known in the art and further described in the following references: Spatola, A. F.
  • peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Peptidomimetic compounds can also contain biological equivalents of amino acids such as the sulfonic and boronic acid analogs of amino acids.
  • the term "normalize,” when used in the context of lipid content in skeletal muscle, refers to a reduction in lipid content in muscle of individuals with abnormally high levels of muscle triglyceride, e.g., individuals who would benefit from a reduction in muscle triglyceride.
  • the term "minimal,” when referring to the effect of a compound on a metabolic response (e.g., increase in blood glucose or lactate levels) of an animal, tissue, or cell refers to an effect of a compound that mediates an increase in lipolysis by stimulating the high affinity receptor, but does not significantly increase the metabolic response due to stimulation of the amylin metabolic receptor.
  • the terms “minimally stimulates vasodilatation” “minimally stmiulates side-effects” “or minimally increases blood lactate” and the like can refer to an agent or amount of agent for which the ratio: [increase in lipolysis]/[increase in blood glucose/elevation of blood lactate/increase in vasodilatation/increase in side-effects, etc.] is higher than the ratio resulting from stimulation by a metabolic amylin receptor saturating amount of CGRP-1 (e.g., 200 nM). Usually the ratio is at least about 2-fold higher, often at least about 4-fold higher, sometimes at least about 10-fold higher). Also encompassed by these terms are agents or doses for which no significant increase in vasodilatation and/or elevation of blood lactate and/or other side-effects is detected (i.e. where the denominator is close to zero).
  • Calcitonin gene-related peptide is a 37 amino acid member of the calcitonin family of peptide hormones. CGRP is present in efferent, acetylcholine containing neurons innervating motor end plates of skeletal muscles where it is released into the synaptic space after nerve stimulation. CGRP is found in humans in at least two predominant forms, called CGRP-1 and CGRP-2 (Table 1).
  • CGRP shares approximately 50% sequence similarity to the protein amylin (Cooper et al., 1987, Proc Natl Acad Sci USA 84:8628-32; Cooper et al., 1994, Endocr Rev. 15:163-201) and the two proteins have overlapping, but different, sets of biological activities (see, e.g., Muff et al., 1995, Eur. J. Endocrinol. 133:17-20).
  • CGRP cardiovascular effects
  • vasodilatation and positive chronotropic and inotropic effects include vasodilatation and positive chronotropic and inotropic effects (Nuki et al., 1993, Biochem Biophys Res Commun 196:245-51; Muff et al., supra; Cooper et al., 1994, Endocr Rev. 15:163-201; Gardiner, 1991, Diabetes 40:948-951; and Takenaga, 1999, Euro. J. Pharma. 367:239-245).
  • CGRP is reported to lead to insulin resistance (see, e.g., Leighton and Cooper, 1988, Nature 335:632-35).
  • insulin resistance should be treated by inhibiting the release or activity of CGRP (see, e.g., U.S. Pat. No. 6,004,961; U.S. Pat. No. 5,641,744).
  • CGRP CGRP
  • elevated plasma amylin and CGRP levels are associated with insulin resistance, and in humans, with obesity and type 2 diabetes.
  • amylin and CGRP have been reported to inhibit glucose uptake by inhibition of glycogen synthesis and to promote glycogenolysis, opposing the actions of insulin as non-competitive, functional antagonists.
  • the elevated circulating lactate from glycogenolysis serves as a substrate for liver gluconeogenesis, causing excessive hepatic glucose production.
  • the present invention is related, in part, to the discovery that CGRP-1 stimulates lipolysis (i.e., cause the breakdown of triacylglycerol [triglycerides]) in skeletal muscle and liver, and rat CGRP-2 was demonstrated to stimulate lipolysis in muscle. This breakdown can be detected as an increase of free fatty acids.
  • dose-response assays in which skeletal muscle was exposed to various amounts of rat CGRP-1 show a biphasic increase in muscle free fatty acid levels, with a first phase observed at a picomolar concentration of CGRP- 1 and a second phase observed at a nanomolar concentration of CGRP-1 or 2.
  • the data indicate that potent lipolysis effects of CGRP-1 on muscle lipid are effected via a high affinity CGRP receptor (EC 50 about 2.6 x 10 "13 M [e.g., range 10 "11 M to 10 "13 M]).
  • a cell that expresses the high affinity receptor is characterized by responding to picomolar concentrations of CGRP-1. This concentration is much lower than those that elicit insulin resistance and inhibition of glycogen synthesis in isolated muscle preparations (Leighton et al., 1988, Nature 335:632-635; Leighton et al., 1989, FEBS Lett 249:357-361).
  • a second phase of lipolysis occurs at higher concentrations of CGRP-1 or 2 (EC 50 about 4.5 x 10 "8 M [e.g., range 10 "9 M to 10 "8 M]) and is mediated by the metabolic amylin receptor, i.e., a single receptor that can transduce signaling by both CGRP and amylin in skeletal muscle, where it can act as a receptor for either peptide (that is, a CGRP/amylin receptor).
  • the metabolic amylin receptor i.e., a single receptor that can transduce signaling by both CGRP and amylin in skeletal muscle, where it can act as a receptor for either peptide (that is, a CGRP/amylin receptor).
  • an agonist of the high affinity CGRP receptor is characterized by a dose-dependent stimulation of lipolysis in skeletal muscle, in which stimulation is mediated by the receptor with which CGRP- 1 interacts with a picomolar EC 50 .
  • An agonist of the metabolic amylin receptor is characterized by stimulation of lipolysis in skeletal muscle mediated by the receptor through which CGRP-1 or 2 interacts with a nanomolar EC 50 , and with which amylin elicits lipolytic effects.
  • a receptor with the characteristics of the metabolic amylin receptor is constituted by co-transfection of the calcitonin receptor (insert-negative form) [Cli ns -] with ramp 1 into either L6 myoblasts or COS-7 cells (see Examples, infra).
  • This receptor constitutes strong specific binding of CGRP, amylin and sCT, and transduction of signals by both CGRP and amylin.
  • High affinity binding sites for CGRP-1 have been described in muscle and liver cells. Galeazza et al., 1991, Peptides 12:585-91 described two CGRP-1 binding sites in rat skeletal muscle membrane preparations, with derived Kd values of approximately 37 pM and 6 nM.
  • Galeazza et al. also described two specific binding sites for CGRP in rat liver membranes, with derived Kd values of approximately 44 pM and 27 nM. Binding of radiolabeled CGRP-1 to membrane fractions of liver nonparenchymal cells (e.g., endothelial, lipid storage, and smooth muscle cells) but not in parenchymal liver cells was reported by Stephens et al., 1991, Diabetes, 40:395-400. Also see Poyner et al., 2002, Pharmacol. Rev. 54:233-46.
  • liver nonparenchymal cells e.g., endothelial, lipid storage, and smooth muscle cells
  • lipolysis can be mediated via the high affinity CGRP receptor, and other discoveries detailed herein, provide new methods and reagents for stimulation of lipolysis and reduction of accumulation of lipid in cells and tissues.
  • the invention provides methods and reagents to effect reduction of lipids in skeletal muscle and liver, and for treatment of diseases characterized by accumulation of lipid in cells and tissues.
  • Accumulation of lipid in skeletal muscle is a key metabolic abnormality underlying the development of insulin resistance. For example, in obesity-related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized towards fat esterification rather than oxidation, and dietary-induced weight loss does not correct this disposition (Simoneau et al., 1999, FASEB J. 13:2051-60).
  • Diseases and conditions characterized by accumulation of lipid in skeletal muscle include insulin resistance and associated conditions, such as those of Syndrome X (e.g., type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis, and thrombosis) and poly cystic ovary syndrome (PCOS).
  • Diseases and conditions characterized by accumulation of lipid in liver include fatty liver (hepatic steatosis). Hepatic steatosis can arise in patients with solvent injury (e.g., from carbon tetrachloride) or chronic excessive alcohol consumption, obese patients and as a result of administration of drugs such as glucocorticoids and synthetic estrogens.
  • the invention provides methods and reagents for treatment of these and other conditions.
  • the invention also provides methods for identification and development of pharmaceutical compositions useful for stimulating lipolysis in muscle and other tissues.
  • the present invention provides methods for treating insulin resistance using an agonist of the high affinity CGRP receptor normally bound by CGRP-1, while mimizing or avoiding the adverse side effects (e.g., vasodilatation; acute cardiovascular effects) that normally accompany administration of high doses CGRP.
  • lipolysis can be achieved, while lninimizing the increase in circulating lactate (e.g., resulting from glycogenolysis) and elevated blood glucose (e.g., resulting from lactate-stimulated hepatic glucose production).
  • lactate e.g., resulting from glycogenolysis
  • blood glucose e.g., resulting from lactate-stimulated hepatic glucose production
  • the present invention provides a method of reducing lipid content in a tissue or cell (e.g., skeletal muscle or liver) by contacting the tissue or cell with an agonist of the high affinity CGRP receptor or the metabolic amylin receptor.
  • a tissue or cell e.g., skeletal muscle or liver
  • the present invention provides a method of reducing lipid content in skeletal muscle or liver tissue or a skeletal muscle or liver cell, by contacting the tissue or cell with an agonist of the high affinity CGRP receptor.
  • a tissue or cell is contacted with an agonist by administration of the agonist to an animal, typically a mammal.
  • the invention provides a method for treating a mammal in need of a reduction in lipid content (for example, of skeletal muscle or liver) to ameliorate symptoms of, or reduce likelihood of developing, conditions associated with muscle fat accumulation, particularly including insulin resistance and "syndrome X" complex (e.g., type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis, and thrombosis), polycystic ovary syndrome, and hepatic steatosis, by administering an agonist of the high affinity CGRP receptor.
  • the agonist is human CGRP, e.g., human CGRP-1.
  • the agonist of the high affinity CGRP receptor does not substantially stimulate the metabolic amylin receptor.
  • the mammal may be a human, a primate, a nonhuman primate, or a nonhuman animal.
  • the invention provides a method of treating a mammal, such as a human, in need of a reduction in lipid content (for example, of skeletal muscle or liver) to ameliorate symptoms of, or reduce likelihood of developing, conditions associated with muscle fat accumulation, by (1) identifying an animal with a condition associated with fat accumulation (e.g., insulin resistance, type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis, thrombosis, polycystic ovary syndrome, or hepatic steatosis) (2) administering an agonist of the high affinity CGRP receptor, and (3) monitoring lipolysis in the animal.
  • the condition is insulin resistance.
  • the condition is type-2 diabetes mellitus.
  • the mammal is insulin resistant but does not have type II diabetes.
  • the mammal is not treated with insulin.
  • Identification of a mammal as suffering from an aforementioned condition is readily accomplished using art-known diagnostic methods (including methods described hereinbelow) but can be accomplished using any identification method known or developed in the future (e.g., gene expression profiling).
  • An exemplary agonist of of the high affinity CGRP receptor is human CGRP-1 and biologically functional variants of human CGRP-1. In an embodiment, administration of the agonist does not substantially stimulate the metabolic amylin receptor.
  • the invention provides the use of an agonist of the high affinity CGRP receptor for the manufacture or formulation of a medicament for reduction of lipid levels in a subject who would benefit from such a reduction.
  • the invention provides the use of an agonist of the high affinity CGRP receptor for the manufacture or formulation of a medicament for treatment of a subject with Syndrome X syndrome, insulin resistance, type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis, and thrombosis.
  • the invention provides pharmaceutical compositions in unit dosage form which contain an agonist of the high affinity receptor as described herein.
  • the unit dosage results, when administered to a mammal such as a human, in a blood or plasma level of agonist that is below the EC 50 of the active agent for the metabolic amylin receptor.
  • a blood or plasma level of agonist of the agent is present between about 10 " 5 M and about 10 "10 M).
  • the plasma level may be below 300 pM (see Examples, infra).
  • Suitable receptor agonists can be any of a variety of agents, as discussed in detail infra, including a naturally occurring CGRP polypeptide (e.g., human CGRP-1 polypeptide), a CGRP polypeptide variant (e.g., a biologically functional variant), or a non-polypeptide agent.
  • CGRP receptor agonist administered to a subject (i.e., in vivo contacting) or ex vivo contacting a tissue or cell with the agonist, can result in a detectable reduction in lipid content and concomitant increase in free fatty acids. It is believed that lipid content is reduced as a consequence of stimulation of lipolysis in cells, e.g., skeletal muscle cells. See, e.g., Example 17, infra. However, applicants do not intend to be bound by any particular mechanism. Thus, while phrase "stimulation of lipolysis” is sometimes used herein for convenience, it is intended to encompass a reduction of lipid levels and increase in free fatty acids in a tissue, without regard to the mechanism of the reduction and increase.
  • CGRP- receptor agonist stimulation of lipolysis can be detected as a change in amount or composition of free fatty acids in a tissue (e.g., skeletal muscle or liver, or blood or blood plasma).
  • a tissue e.g., skeletal muscle or liver, or blood or blood plasma.
  • the level of stimulation of lipolysis is an increase in tissue or blood free fatty acid levels of at least about 2-fold, often at least about 4-fold, and sometimes at least about 6-fold or more over baselines (levels in the absence of the agonist).
  • Methods for detecting a change in free fatty acid levels are well known. For example, as described in Example 2, infra, gas chromatographic analysis of muscle tissue (e.g., obtained from biopsy or tissue culture) can be conducted. Other methods include HPLC procedures for preparative scale separations of particular fatty acids for structural or metabolic studies.
  • Tissue long chain CoA can be measured by solvent extraction of long chain CoA from tissues, phase separation, and purification by reverse phase high performance liquid chromatography.
  • the invention provides the use of an agonist of the high affinity CGRP receptor for the manufacture or formulation of a medicament for reduction of lipid levels in a subject who would benefit from such a reduction.
  • the invention provides the use of an agonist of the high affinity CGRP receptor for the manufacture or formulation of a medicament for treatment of a subject with Syndrome X syndrome, insulin resistance, type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis, and thrombosis.
  • the invention provides a method of treating a mammal in need of a reduction in lipid content comprises administering an agent that is an agonist of the metabolic amylin receptor, but not the high affinity receptor, to stimulate lipolysis in a mammal, methods of treating mammals in need of treatment for lipolysis reduction by administering such an agonist of the metabolic receptor, and pharmaceutical compositions comprising such an agonist to achieve the result.
  • the agonist is rat CGRP-2.
  • the agonist is a receptor stimulating varient of CGRP.
  • the agonist is not amylin.
  • the agonist is amylin.
  • Agonists of the high affinity CGRP receptor used in the practice of the invention include a variety of types of compounds, including peptides, peptide mimetics and nonpeptide compounds. Suitable agonists are described below and/or can be identified using methods described herein (and/or by methods that will be apparent to one of ordinary skill guided by this disclosure). Agonists of the high affinity CGRP receptor stimulate lipolysis in skeletal muscle. Preferred agonists and/or dosages result in preferential stimulation of the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • PREFERENTIAL STIMULATION As noted herein, certain tissues (e.g., skeletal muscle) express both the high affinity receptor and the metabolic amylin receptor, and certain compounds, such as rat CGRP-1, are agonists of both the high affinity CGRP receptor and the metabolic amylin receptor. As noted above, lipolysis can be mediated by both receptors. However, as disclosed herein, it will often be desirable to preferentially stimulate the high affinity "Preferential stimulation" has the normal meaning of the term and can be described (or detected) in a variety of ways.
  • Preferential stimulation occurs when the agonist of the high affinity receptor has an EC 50 for the high affinity receptor that is lower than the EC 50 for the metabolic receptor, and the dose or concentration of the agonist is below the EC 50 for the metabolic amylin receptor but at or above the EC 50 for the high affinity receptor.
  • "preferential stimulation” is accomplished when the high affinity receptor agonist does not cause any detectable stimulation of the metabolic amylin receptor (e.g., at any dose, or , alternatively, at any dose less than 10 microM, or any dose less than 10 mM).
  • the agonist of the high affinity CGRP receptor is administered in an amount that results in preferential stimulation of the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • preferential stimulation can be achieved, e.g., by adjusting the dose or formulation of an agonist, or due to the properties of the agonist.
  • preferential stimulation of the high affinity CGRP receptor compared to the metabolic amylin receptor is accomplished by contacting the cell with an agent that causes preferential stimulation of the high affinity receptor.
  • the agent is an agonist of the high affinity CGRP receptor but does not substantially agonize the metabolic amylin receptor.
  • the agent has an EC 5 o for effecting lipolysis stimulation via the metabolic amylin receptor significantly greater than the EC 50 of the agent for effecting lipolysis stimulation via the high affinity CGRP receptor.
  • contacting skeletal muscle with the agent results in greater production of FFA (e.g., as measured by appearance of FFA in tissue or plasma) due to the lipolysis-stimulating effects of the high affinity receptor than due to the lipolysis- stimulating effects of the metabolic amylin receptor.
  • one way to determine whether an effect of an agent is a result of stimulation of the high affinity CGRP receptor is by comparing the effect of the agent with the effects of amylin (which has a similar dose response curve as CGRP) which is known to act via the metabolic receptor.
  • the agent with an EC 50 for effecting lipolysis stimulation via the metabolic amylin receptor significantly greater than the EC 50 of the agent for effecting lipolysis stimulation via the high affinity CGRP receptor has an EC 50 for the high affinity CGRP receptor at least 10-fold less than for the metabolic amylin receptor, and more often at least about 100-fold less, at least about 500-fold less, at least about 1000-fold less, at least about 5000-fold or at least about 10,000-fold less. Most often, however, the EC 50 for the high affinity receptor is at least about 10 5 -fold less, 10 6 -fold less, 10 7 -fold less, or 10 8 -fold less.
  • the ratio for an agent with a significantly greater EC 50 for stimulation via the high affinity receptor is usually at least about 10, often at least about more than about 10 2 , more than 5 x 10 2 , more than about 10 3 , more than 5 x 10 3 , more than about 10 4 , more c Q than about 10 , more than about 10 , more than about 10 , or more than about 10 .
  • Methods for determining the EC 50 of a compound are known generally, and are described hereinbelow.
  • the agonist of the high affinity CGRP receptor does not have any significant or any detectable stimulatory activity for the metabolic amylin receptor when administered or contacted with a cell or tissue.
  • One measure of significant stimulatory activity for the metabolic amylin receptor is a significant increase in blood lactate and blood glucose resulting from infusion of the agonist into an animal (e.g,. rat) relative to a control animal infused with saline or other non active composition. Significance is detemiined by routine methods such as the mean and standard error inherent in the measurements. Significance between control versus treated means can be tested statistically using standard analyses such as Student's t-test.
  • assays are conducted using an n of >1, e.g., at least 7 for greater statistical power.
  • Assays for increased lactate production and antagonism of insulin-stimulated glucose uptake in skeletal muscle via the metabolic amylin receptor are well known (see, e.g., Cooper, 1994, Endocr Rev. 15:163-201; Young et al, 1993, FEBS Lett. 334 (3) 317-321; and Young et al, "Amylin Activity Assays" U.S. Patent 6,048,514).
  • assays can be carried out using human cells or tissues, cells or tissues from other mammals (e.g., mice or rats), cell lines, and recombinant cells. Assays include cell-based assays; ex vivo assays (e.g., in isolated rat soleus muscle), and whole animal studies. For example, cell-based assays can be carried out using the cell-based recombinant receptor described in the Examples, infra.
  • isolated rat soleus muscle is used. Rat soleus muscle contains both the high affinity CGRP receptor and the metabolic amylin receptor and isolated rat soleus muscle is particularly useful to distinguish differential effects on the different receptors.
  • the soleus muscles can be derived from animals that have been fed a high fat or normal diet and dose-response curves for a compound prepared in which (i) assays reflective of processes that can occur through both the high affinity CGRP receptor and the metabolic amylin receptor (e.g., measurement of muscle free fatty acid and triglyceride content), and (ii) reflective of processes occurring through the metabolic amylin receptor (e.g., measurement of muscle glycogen content and [ 14 C]-glucose incorporation into glycogen in the presence of maximally-stimulating insulin) are made.
  • assays reflective of processes that can occur through both the high affinity CGRP receptor and the metabolic amylin receptor e.g., measurement of muscle free fatty acid and triglyceride content
  • reflective of processes occurring through the metabolic amylin receptor e.g., measurement of muscle glycogen content and [ 14 C]-glucose incorporation into glycogen in the presence of maximally-stimulating insulin
  • An agent, dose or formulation that elicits effects indicating stimulation of the high affinity CGRP receptor but not effects indicative of stimulation specific to the metabolic amylin receptor are considered agonist of the high affinity CGRP receptor that do not substantially agonize the metabolic amylin receptor.
  • the effects on the high affinity and metabolic receptors are distinguished by selective antagonists of the high affinity and/or metabolic receptor.
  • An agent or treatment that does not cause a significant change in an activity of the metabolic amylin receptor e.g., an increase in receptor-mediated lipolysis
  • control tissues, cells or animals e.g., not exposed to the agonist
  • the agonist of the high affinity receptor is administered to a mammal (or contacted with an isolated tissue) at a dose sufficient to stimulate the skeletal muscle high affinity CGRP receptor in the host without substantial stimulation of the low affinity skeletal muscle metabolic amylin receptor.
  • the effect of an agent that stimulates both the high affinity receptor and the metabolic amylin receptor at high concentration can be modulated to maximize relative to any stimulation of the metabolic amylin receptor by adjusting the dose administered (for example, adjusting the formulation such that only a measured amount of active compound is released and available to interact with the receptor).
  • dose administered in intended to include ex vivo contacting of tissues with the agent.
  • a preferred dosage range for administration to a mammal (e.g., human) of an agonist of the high affinity receptor is the smallest dose that results in a reduction in skeletal muscle or liver lipid of at least about 15%, preferable at least about 25%, more preferably at least about 30%, even more preferably at least about 50%, or more, such as at about 75% (e.g. as measured by a change in free fatty acid or triglyceride levels).
  • Another preferred dosage range for administration to a mammal (e.g., human) of an agonist of the high affinity receptor is an amount that results in stimulation of lipolysis in skeletal muscle of a mammal without stimulating, or minimally stimulating, vasodilatation.
  • This amount can be determined any number of ways, such as by (i) conducting dose-response assays by administering a plurality of different doses of a CGRP receptor agonist formulation to test mammals (e.g., by I.V., I.P., oral, or other routes); (ii) measuring the effect of each dose on lipolysis in muscle of the test mammal and measuring the effect of each dose on vasodilatation in the test mammal, thereby creating dose-response data for lipolysis and vasodilatation; and, (iii) determining from the dose-response data a dose of the CGRP receptor agonist formulation that increases lipolysis but does not significantly increase vasodilatation, or only minimally increases vasodilatation, in the mammal.
  • Vasodilatation can measured using any of a variety of assays (see, e.g., Nuki et al., 1993, Biochem Biophys Res Commun 196:245-51). In humans, vasodilatation can be monitored by measurement of systolic blood pressure and mean arterial blood pressure.
  • Another preferred dosage range for administration to a mammal (e.g., human) of an agonist of the high affinity receptor is an amount that results in stimulation of lipolysis in skeletal muscle of a mammal without stimulating, or only minimally stimulating, an increase in levels of glucose and/or lactate in blood (i.e., a statistically significant increase over baseline levels).
  • This amount can be determined any number of ways, such as by (i) conducting dose-response assays by administering a plurality of different doses of a CGRP receptor agonist formulation to test mammals (e.g., by I.V., I.P., oral, or other routes); (ii) measuring the effect of each dose on lipolysis in muscle of the test mammal and measuring the effect of each dose on glucose and/or lactate levels in the test mammal, thereby creating dose-response data for lipolysis and glucose/lactate levels; and, (iii) determining from the dose-response data a dose of the CGRP receptor agonist formulation that increases lipolysis but does not increase, or only minimally increases, glucose and/or lactate levels in the mammal.
  • Blood glucose and lactate can be measured using routine methods, such as through immobilized enzyme chemistries such as is found in a standard glucose analyzer (glucose oxidase, L-lactate oxidase, Analyzer model 2300-STAT, Yellow Springs Instruments, Yellow Springs, Ohio). Briefly, the substrate is oxidized as it enters the enzyme layer, producing hydrogen peroxide, which passes through cellulose acetate to a platinum electrode where the hydrogen peroxide is oxidized. The resulting current is proportional to the concentration of the substrate. Also see, for example, Ye et al., 2001, Diabetes, 50: 411-417; Ellis et al., 2000, Am. J. Phys. 279:E554-E560.
  • CGRP-1 polypeptide e.g., human CGRP-1
  • a pharmaceutical formulation that results in a plasma or serum concentration of CGRP-1 in the EC 50 range for the high affinity CGRP receptor (e.g., range 10 "11 M to 10 "13 M) but below the EC 50 for the metabolic amylin receptor (e.g., range 10 "9 M to 10 "8 M) is particularly useful.
  • an exemplary dose results in a plasma or serum level of between about 10 "15 M and about 10 "10 M, between about 10 "15 M and about 10 "11 M, or between about 10 "15 M and about 10 "12 M.
  • Plasma and serum levels of compounds can be measured by routine means, such as ELISA, RIA, spectroscopy, enzymatic assays, or other methods).
  • a variety of compounds may agonize the high affinity CGRP receptor, including polypeptides and peptidomimetics, and non-polypeptide compounds such as small organic molecules (e.g., molecular weight ⁇ 1000).
  • the high affinity CGRP receptor agonist is a polypeptide.
  • the polypeptide has a sequence identical to, or with significant sequence similarity to a naturally occurring CGRP-1 polypeptide (e.g., a human CGRP-1 polypeptide or a homolog from rat, pig, cow, rabbit, chicken, salmon, or other species; see Cooper et al., 1994, Endocr Rev. 15:163-201).
  • a specified polypeptide has significant sequence similarity to a naturally occurring CGRP-1 polypeptide when the specified polypeptide comprises a sequence of amino acids that, when aligned for optimal match, corresponds to at least about 75%, sometimes at least about 80%, and sometimes at least about 90% of the residues of the naturally occurring CGRP-1 polypeptide (e.g., human).
  • the sequence of the receptor agonist differs from CGRP-1 (e.g., human CGRP-1) only by conservative substitutions.
  • the high affinity CGRP receptor agonist is a biologically functional variant of a CGRP-1 polypeptide, such as a polypeptide or peptide analog (such as a peptidomimetic).
  • Polypeptides that are biologically functional variants of CGRP-1 are characterized by the following properties: (i) they stimulate lipolysis in skeletal muscle, (ii) they have an EC 50 for effecting lipolysis stimulation via the metabolic amylin receptor significantly greater than the EC 50 for effecting lipolysis stimulation via the high affinity CGRP receptor, i.e., they can preferentially stimulate the high affinity CGRP receptor), and usually they (iii) comprise a sequence of amino acids that, when aligned for optimal match, correspond along the length of the variant to a naturally occurring CGRP-1 sequence, e.g., human CGRP-1 sequence, at least about 60%, sometimes at least about 75%, sometimes at least
  • variants can be designed and tested for agonist activity.
  • Conventional methods for mutagenesis e.g., site-directed mutagenesis, alanine scanning and analysis using methods described herein or known in the art, can be used to identify biologically functional variants of CGRP-1.
  • the invention provides a method of making a non-naturally occurring agonist of the high affinity CGRP receptor useful in preparation of a therapeutic agent by (i) obtaining a sequence of a naturally occurring CGRP polypeptide (e.g., human CGRP-1); (ii) modifying at least one amino acid residue by substitution or deletion to create a CGRP variant; (iii) testing the ability of the variant to stimulate one or more activities mediated by the high affinity CGRP receptor (e.g., lipolysis- stimulating activity in skeletal muscle); and (iv) identifying a variant that can preferentially agonize the high affinity CGRP receptor compared to the metabolic amylin receptor as a non-naturally occurring agonist of the high affinity CGRP receptor useful in preparation of a therapeutic composition.
  • a naturally occurring CGRP polypeptide e.g., human CGRP-1
  • modifying at least one amino acid residue by substitution or deletion to create a CGRP variant
  • biologically functional variants of CGRP-1 that act as agonists of the high affinity CGRP-1 receptor include such an amino acid ring structure, or an equivalent (e.g., a nonpeptide structural homolog), and/or are amidated.
  • the agonist is a polypeptide having the fonnula of a reported consensus sequence for the CGRP family: Xxx Cys Xxx Thr Ala Thr Cys Val Thr His Arg Leu Ala Xxx Xxx Leu Xxx Arg Ser Gly Gly Xxx Xxx Xxx Asn Phe Val Pro Thr Xxx Val Gly Xxx Xxx Ala Phe, where Xxx is any amino acid. See, Cooper et al, Endocr Rev. 1994 15:163-201.
  • the agonist is, or corresponds to, a fragment of a CGRP polypeptide (e.g., at least 6 residues, more often at least about 20, 25, 30 or 35 residues).
  • the agent has a sequence of a chimeric CGRP polypeptide in which one or more amino acids in a naturally occurring sequence from one species (e.g., human) is replaced with a different amino acid found the corresponding sequence of a CGRP-1 from one or more different species (e.g., rat).
  • CGRP peptides can be prepared by routine synthetic or recombinant methods, using known sequences for these proteins. Recombinant techniques and other methods useful in the practice of the present invention are known in the art and are described in, for example, Sambrook and Russel (2001) MOLECULAR CLONING: A LABORATORY MANUAL (3rd Edition) Cold Spring Harbor Laboratory Press; Ausubel et al. (1987) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (as supplemented through 2001), John Wiley & Sons, New York.
  • CGRP polypeptides De novo chemical synthesis of polypeptides is well known and can be used to prepare CGRP polypeptides (see e.g., Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; and Horn et al, 1980, Nucleic Acids Res. Symp. Ser., 225-232).
  • peptide synthesis can be performed using various solid-phase techniques (Roberge, et al., 1995, Science 269:202), including automated synthesis (e.g., using the Perkin Elmer ABI 431 A Peptide Synthesizer in accordance with the instructions provided by the manufacturer).
  • the newly synthesized peptide can be substantially purified, for example, by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co, New York NY [1983]).
  • the amino acid sequence of the CGRP polypeptide, or any part thereof may be altered during direct synthesis and or combined using chemical methods with sequences from other proteins (particularly sequences from homologous CGRP polypeptides from the same or other animal species), to produce a variant polypeptide of the invention.
  • CGRP polypeptides can be isolated from natural sources.
  • the agonist(s), or test compound(s) or agent(s) is a compound other than CGRP.
  • the invention provides a method for determining whether an agent useful for stimulating lipolysis in a mammal by determining whether the agent is an agonist of the high affinity CGRP receptor. In one contemplated embodiment of this method, the agent is not CGRP.
  • the agonist is other than a CGRP -related polypeptide.
  • the agonist can be a polypeptide of unrelated sequence or, alternatively, a non-polypeptide molecule.
  • Exemplary non-polypeptide agonists may be compounds such as carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; lipids or phospholipids; fatty acids; steroids; dipeptides, amino acid analogs, and organic molecules, e.g., small molecules.
  • the agonist is other than CGRP-1 of a human or other than a CGRP-1 polypeptide of a mammal.
  • a polypeptide or non-polypeptide agonist is prepared using rational drug design methods (e.g., using an integrated set of methodologies that include structural analysis of target molecules, synthetic chemistries, and advanced computational tools). See, e.g., Kim et al., 2000, Comb Chem High Throughput Screen. 3:167-83 and Coldren, 1997, Proc. Natl. Acad. Sci. USA 94:6635-40:
  • the agonist is a compound identified according to a screening method described hereinbelow.
  • the invention provides a method of preparing a pharmaceutical composition by preparing the agonist as above, and combining the agonist with a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • the invention provides methods for stimulation of lipolysis by stimulation of either the high affinity CGRP receptor, the metabolic amylin receptor, or both, using CGRP-1 or polypeptides that are metabolic receptor stimulating variants of CGRP-1, based on the discovery that CGRP-1 binding to the metabolic amylin receptor stimulates lipolysis.
  • CGRP-1 binding to the metabolic amylin receptor stimulates lipolysis.
  • a further step of monitoring a change in the level of lipolysis in a tissue of the mammal e.g., by measuring the amount of free fatty acids in muscle, liver, blood or other tissue of the mammal or isolated tissue.
  • Metabolic receptor stimulating variants of CGRP-1 are characterized by the following properties: (i) they stimulate lipolysis in skeletal muscle, and (ii) they comprise a sequence of amino acids that, when aligned for optimal match, correspond along the length of the variant to a naturally occurring CGRP-1 sequence, e.g., human CGRP-1 sequence, at least about 60%, sometimes at least about 75%, sometimes at least about 80%, and sometimes at least about 90% of the residues.
  • the invention provides a method of stimulating lipolysis in skeletal muscle or liver of a mammal by contacting the skeletal muscle or liver with CGRP-1 or a metabolic receptor stimulating variant thereof.
  • the invention provides a therapeutic regimen including (i) adrninistering a CGRP-1 polypeptide, a biologically function variant thereof, or a metabolic receptor stimulating variant thereof to a mammal suffering from or susceptible to a condition characterized by accumulation of lipid in skeletal muscle, and (ii) monitoring lipolysis in the mammal.
  • a therapeutically effective amount of an agonist of the high affinity CGRP receptor is administered to a subject (e.g., patient or animal) who would benefit from a reduction in tissue lipid content (e.g., skeletal muscle lipid content).
  • tissue lipid content e.g., skeletal muscle lipid content
  • an agonist of the high affinity CGRP receptor e.g., a CGRP-1 polypeptide
  • tissue lipid content e.g., skeletal muscle lipid content
  • tissue lipid content e.g., skeletal muscle lipid content
  • tissue lipid content e.g., skeletal muscle lipid content
  • tissue lipid content e.g., skeletal muscle lipid content
  • the dosage ranges for the administration of the agents of the invention are those large enough to produce the desired effect (e.g., reduction in lipid content or amelioration of symptoms or progression of the condition).
  • the invention provides an agonist of the high affinity CGRP receptor in a unit dosage form for administration to patients.
  • unit dosage form refers to a composition intended for a single administration to treat a subject suffering from a disease or medical condition.
  • Each unit dosage form typically comprises each of the active ingredients of this invention plus pharmaceutically acceptable excipients.
  • Examples of unit dosage forms are individual tablets, individual capsules, bulk powders, liquid solutions, suppositories, emulsions or suspensions. Treatment of the disease or condition may require periodic administration of unit dosage forms, for example: one unit dosage form two or more times a day, one with each meal, one every four hours or other interval, or only one per day.
  • oral unit dosage form indicates a unit dosage form designed to be taken orally. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers.
  • the unit dosage form of the invention contains a therapeutically effective dose of the receptor agonist. In an embodiment, administration of the unit dosage form results in a level of agonist in the mammal to preferentially stimulates the high affinity receptor (compared to the metabolic amylin receptor).
  • the agonist of the high affinity CGRP receptor e.g., CGRP-1 protein or biologically functional variant thereof
  • the agonist of the high affinity CGRP receptor is administered at a concentration that does not saturate binding to the metabolic amylin receptor (e.g., as monitored through effects known to occur through the metabolic amylin receptor, such as increases in blood or plasma lactate and glucose levels).
  • the dose results in only minimal increase (if any) in blood or plasma lactate and glucose levels or vasodilatation.
  • a dose or formulation of an agonist of the high affinity CGRP receptor that stimulates lipolysis in skeletal muscle of a mammal without or only minimally eliciting an undesired side-effect in the mammal can be determined in a variety of ways.
  • an increased level can refer to an increase to a predetermined level (e.g., a designated threshold level of the sideeffect).
  • a predetermined level e.g., a designated threshold level of the sideeffect.
  • One method for making such determination involves ) conducting dose-response assays by (a) administering a plurality of different doses (or formulations) of the CGRP receptor agonist to test mammals; and (b) measuring the effect of each dose or formulation on lipolysis in a tissue of the test mammal and measuring the effect of each dose on the side-effect, thereby creating dose-response data for lipolysis and the side-effect; and, (ii) determining from the dose-response data a dose of the CGRP receptor agonist formulation that increases lipolysis but does not elicit the side-effect.
  • the effect of each dose or formulation on lipolysis is determined by measuring free fatty acid levels in a tissue of the animal. Analogous methods can be carried out using isolated tissues in place of whole animals. Further, based on the guidance herein, it will be within the ability of one of ordinary skill to dete ⁇ nining the desired dose using any of a variety of methods.
  • the agonist doses for non-polypeptide compounds would fall in a concentration of from 10 "16 M to 10 "5 M (e.g., as measured in one or more of muscle, blood, serum or plasma).
  • CGRP-1 polypeptide e.g., human CGRP-1
  • a dose that results in a plasma or serum concentration of CGRP-1 in the EC 50 range for the high affinity CGRP receptor (10 "u M to 10 "13 M) but below the EC 50 for the metabolic amylin receptor (10 "9 M to 10 “8 M) is particularly useful.
  • an exemplary dose results in a serum or plasma level of between about 10 "15 M and about 10 "10 M. In one embodiment, the serum level is less than 300 pM.
  • the amount of an agent administered to an animal to achieve a desired level or concentration of agent will depend on a number of factors well know to practicioners, such as compound half-life (e.g., serum half-life), and the frequency and mode of administration.
  • the dose of a CGRP-1 polypeptide is administered in the range from 20 picograms to 1 gram, more often between 3 nanograms and 50 micrograms daily.
  • the unit dosage in some cases daily dosage is less than about 10 micrograms, less than about 1 microgram, less than about 100 nanograms, less than about 10 nanograms, less than about 1 nanogram, less than about 100 picograms, or less than about 10 picograms.
  • the invention also provides a composition containing an agonist of the high affinity CGRP receptor combined with a pharmaceutically acceptable excipient.
  • the agent and excipient are formulated to selectively activate a high affinity CGRP receptor without activating an amylin receptor.
  • the agent and excipient are formulated to selectively stimulate lipolysis without, or only minimally, stimulating adverse side effects such as vasodilatation, or elevation of blood glucose or lactate levels.
  • the CGRP polypeptide or other agonist can be formulated or coadministered with other active agents (e.g., agents that alone or in combination CGRP, reduce lipid, free fatty acid, and/or long chain CoA levels). It is contemplated that, in an embodiment, the agonist of the present invention is not coadministered with insulin. In one aspect, the agonist of the high affinity receptor is admininstered with an agent that inhibits activation of the metabolic amylin receptor but not the high affinity receptor (e.g., an anti-receptor antibody). [0125] Agonists (or antagonists) used in the practice of the invention can be directly administered to the host to be treated. Administration is optionally under sterile conditions.
  • active agents e.g., agents that alone or in combination CGRP, reduce lipid, free fatty acid, and/or long chain CoA levels. It is contemplated that, in an embodiment, the agonist of the present invention is not coadministered with insulin. In one aspect,
  • Formulations typically comprise at least one active ingredient together with one or more acceptable carriers thereof.
  • Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject.
  • Therapeutic formulations can be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al (eds.) (1990) GOODMAN AND GlLMAN'S: THE PHARMACOLOGICAL
  • the compounds of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant
  • parenteral e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant
  • inhalation spray nasal, vaginal, rectal, sublingual, or topical routes of administration
  • nasal, vaginal, rectal, sublingual, or topical routes of administration may be formulated, alone
  • agonist can be administered to patients in the form of controlled delivery formulations.
  • suitable controlled delivery systems are known, including forms suitable for oral, parenteral, and other routes of administration. See, e,g, Bogner et al., 1997 U. S. Pharmacist 1997;22(Suppl.):3-12; and GUIDANCE FOR INDUSTRY. EXTENDED RELEASE ORAL DOSAGE FORMS: DEVELOPMENT, EVALUATION, AND APPLICATION OF THE IN VITRO/IN VIVO CORRELATIONS. Rockville, MD: Center for Drug Evaluation and Research, Food and Drug Administration, 1997.
  • Excipients employed in the manufacture of drug delivery systems are described in various publications known to those skilled in the art (for example, see: Kibbe, E. H. HANDBOOK OF PHARMACEUTICAL EXCIPIENTS, 3rd Ed., American Pharmaceutical Association, Washington, 2000, 665 pp.).
  • the USP provides many examples of modified-release oral dosage forms (for example, see: The United States Pharmacopeia 23/National Formulary 18, The United States Pharmacopeial Convention, Inc., Rockville MD, 1995).
  • This publication also presents general chapters and specific tests to determine the drug release capabilities of extended- release and delayed-release tablets and capsules.
  • the agonist is administered in conjunction with a program of exercise, to enhance exercise-mediated breakdown of triglycerides in a subject.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to the known art using those 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.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition. In some embodiments, daily or weekly administration of the agonist is contemplated.
  • a CGRP receptor agonist is administered to a mammal and the mammal is monitored for stimulation of lipolysis or reduction in lipid content in the mammal or in a tissue of the mammal (e.g., in skeletal muscle of the mammal).
  • Lipolysis is usually monitored by detecting a change in amount or composition of free fatty acids in a target tissue (e.g., skeletal muscle, blood, blood plasma).
  • a target tissue e.g., skeletal muscle, blood, blood plasma.
  • acyl CoA oxidase-based colorimetric assay Wako Pure Chemical Industries, Osaka, Japan. Plasma triglycerides can be measured using a colorimetric assay (Triglyceride Procedure 336, Sigma Diagnostics). Recent evidence also suggests that long-chain acyl CoA esters may serve as markers of lipid metabolism and insulin sensitivity in rat and human muscle (see Bronwyn et al., 2000, Am. J. Phys. 279: E554 - E560). Tissue long chain CoA (e.g.
  • derived from muscle biopsies can be measured by measured by solvent extraction of long chain CoA from tissues, phase separation, and purification by reverse phase high performance liquid chromatography (see for example, Bronwyn et al., 2000, Am. J. Phys. 279:E554-E560).
  • new methodologies for the measurement of intramuscular triglyceride content may be used. These include non-invasive methods such as computed tomography and nuclear resonance spectroscopy (see for example, Kelley et al, 1991, J. Clin. Nutr. 54: 509-515; Krssak et al, 1999, Diabetologia 42: 113-116; Jacob et al., 1999, Diabetes, 48: 1113-1119).
  • a CGRP receptor agonist is administered to a mammal and vasodilatation in the mammal is measured.
  • CGRP-1 and CGRP-2 are potent vasodilators (see, e.g., Muff et al., 1995, Eur. J. Endocrinol. 133:17-20; Nuki et al., supra).
  • the skeletal muscle lipolysis-stimulating effects of CGRP-1 can be at least partially uncoupled from the vasodilatation effects, e.g., by administration of an agent of a type, formulation, or dose that agonizes the high affinity receptor but does not agonize (or minimizes activation of) the metabolic amylin receptor.
  • the agent is administered to a mammal and vasodilatation in the mammal is measured.
  • vasodilatation can be monitored by measurement of systolic blood pressure and mean arterial blood pressure.
  • a CGRP receptor agonist is administered to a mammal and blood glucose production and/or lactate levels are measured.
  • lipolysis mediated by the high affinity receptor can be achieved without the undesirable effects mediated by the metabolic amylin receptor (e.g., increased blood glucose levels)
  • a CGRP receptor agonist is administered to a mammal and the mammal is monitored for alleviation of symptoms of a disorder associated with muscle fat accumulation.
  • the course of a disease is monitored at least long enough to detennine whether there is a reduction in severity, symptoms, or progression of the condition .
  • a CGRP receptor agonist is administered to a mammal (e.g., a mammal in need of lipolysis stimulation) and one or more disease or physiological conditions in the mammal are monitored.
  • a CGRP receptor agonist is administered to a mammal, and the mammal is monitored for the effect of administration of the agonist on insulin resistance.
  • Insulin resistance in an animal can be assessed by any of a variety of methods known in the art.
  • insulin resistance can be monitored using an oral glucose tolerance test or OGTT (see, e.g., Bergman et al., 1985, Endocrinology Review 6:45-86).
  • OGTT oral glucose tolerance test
  • individuals with 75 gram, 2 hour OGTT level greater than 140 mg/dl are considered insulin resistant, and individuals a level less than 120 mg/dl are considered normal.
  • Insulin resistance is also be monitored using a steady state blood glucose test (see, e.g., Reaven et al, 1979, Diabetologia 16:17-24). Individuals with SSPG mean greater than 180 mg/dl are usually considered a insulin resistant; individuals with SSPG mean less than 150 mg/dl are considered normal. Other methods include meas rement of HbAlc and C-peptide as markers for insulin resistance (del Prato, 1999, Drugs 58:Supp 1:3-6; Ferranini et al., 1998, Hypertension 16:895-906).
  • any of the above-mentioned monitoring activities can be carried out in combinations; thus, for example, in one embodiment, both vasodilatation and insulin resistance are monitored in the mammal to which the agonist is administered.
  • a condition e.g., insulin sensitivity/resistance
  • effect of agonist administration e.g., lipolysis
  • measurements can be made prior to administration of the agent (e.g., to generate a baseline) and/or at one or more time points after administration (or, in the case of multiple administration, after one or more administrations).
  • the agomst is administered to an individual diagnosed with insulin resistance.
  • the individual is diagnosed as suffering from insulin resistance, but not diagnosed as diabetic (e.g., suffering from type II diabetes).
  • CGRP CGRP receptor agonists
  • a mammal can be administered an amount of CGRP or other agonist sufficient to stimulate lipolysis (and thereby reduce muscle fat accumulation) without eliciting, or only minimally eliciting, increased vasodilatation.
  • the invention provides a method for determining whether an agent is or is not useful for stimulating lipolysis in a mammal or mammalian cell, e.g., in skeletal muscle in a mammal, by determining whether the agent is an agonist of a CGRP receptor.
  • agents that preferentially activate the high affinity CGRP receptor are identified. As noted above, such agents are useful in making therapeutic compositions and in treatment of diseases and conditions.
  • preferential stimulation of the high affinity receptor can be determined in a variety of ways, including by comparing the EC 50 of the agent for effecting a response mediated by the metabolic amylin receptor (e.g., an effect on carbohydrate metabolism) and the EC 50 of the agent for effecting a response mediated by the high affinity CGRP receptor (an increase in free fatty acids in skeletal muscle tissue or a skeletal muscle cell).
  • the metabolic amylin receptor e.g., an effect on carbohydrate metabolism
  • the EC 50 of the agent for effecting a response mediated by the high affinity CGRP receptor an increase in free fatty acids in skeletal muscle tissue or a skeletal muscle cell.
  • the identification and screening methods described herein can be used to screen a single compound or a plurality of compounds for therapeutically useful activity.
  • the plurality may be at least 3 agents, more often at least 25, even more often at least 50, usually at least 100, and may involve screening a very large number (>1000) of agents.
  • Screens may be high-throughput and/or may involve assays carried out sequentially or simultaneously (including beginning the assay for one agent after starting but before completing the assay for another agent).
  • Any suitable assay can be used for identifying preferential activation including (but not limited to) identification on the basis of the EC 50 of the agent for stimulating lipolysis via the high affinity receptor (measured, for example, by a reduction in tissue triacylglceride content or increase in free fatty acid) compared to the EC 5 o (if any) of the agent for stimulating lipolysis via the metabolic amylin receptor and/or identification using other assays described herein (see, e.g., ⁇ 11(B) and examples). It will be recognized that these assays can be used to screen multiple agents, for example, a library of compounds, e.g., in a high-throughput format.
  • Screening assays of the invention can be conducted, without limitation, using animals, in vitro tissue or organ culture (e.g., isolated skeletal muscle), cells or cell lines, for example, to determine whether the compound is useful for reduction in lipid content in an animal, cell or tissue. Alternatively, the agonist is administered to an isolated cell line.
  • the invention provides a method of determining whether an agent is useful for stimulating lipolysis in a mammal by (i) identifying at least one agent, and usually a plurality of agents, that bind the high affinity CGRP receptor (e.g., using CGRP-1 displacement assays); and (ii) selecting an agent that preferentially stimulates the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • the invention provides a method of determining whether an agent is useful for stimulating lipolysis in a mammal by (i) identifying at least one agent, and usually a plurality of agents, that stimulate lipolysis in a tissue expressing the high affinity CGRP receptor; and, (ii) selecting an agent from (i) that preferentially stimulates the high affinity CGRP receptor compared to the metabolic amylin receptor.
  • the method involves (i) at least one agent, and usually a plurality of agents, that bind the high affinity CGRP receptor; and, (ii) selecting an agent from (i) that preferentially stimulates the high affinity receptor compared to the metabolic amylin receptor.
  • the tissue is skeletal muscle (e.g., from rat)
  • the method involves (i) at least one agent, and usually a plurality of agents, that bind the high affinity CGRP receptor; and, (ii) selecting an agent from (i) that has an EC 50 for effecting a response mediated by the metabolic amylin receptor which is higher than the EC 50 of the agent for effecting a response mediated by the high affinity CGRP receptor.
  • the tissue is skeletal muscle (e.g., from rat).
  • the invention provides a method of determining whether an agent is useful for stimulating lipolysis in a mammal on the basis of the EC 50 of the agent for effecting a response mediated by the metabolic amylin receptor and the high affinity CGRP receptor, usually lipolysis, as described hereinabove.
  • the EC 50 of the agent for effecting a response mediated by the metabolic receptor is at least about 10-fold higher, at least about 100-fold, at least about 1000-fold and preferably at least about 10,000-fold higher, at least about 100,000-fold higher, or at least about 1,000,000-fold higher than the EC 50 for effecting the response via the high affinity receptor.
  • soleus muscle e.g., isolated soleus muscle
  • lipolytic activity refers to the ability of an agent to stimulate lipolysis in a cell or tissue.
  • Soleus muscles can be derived from animals that have been fed a high fat or normal diet and dose-response curves for a compound prepared in which (i) assays reflective of processes that can occur through both the high affinity CGRP receptor and the metabolic amylin receptor (e.g., measurement of muscle free fatty acid and triglyceride content), and (ii) reflective of processes occurring through the metabolic amylin receptor (e.g., measurement of muscle glycogen content and [ 14 C]-glucose incorporation into glycogen in the presence of maximally-stimulating insulin) are made.
  • the EC 50 values can be determined from the dose-dependent curves.
  • the dose of the compound will be chosen from results of (i) and (ii). Specifically of interest will be the concentration of compound that elicits metabolic effects in (i) but not in (ii). In the comparison of different compounds, this concentration will be a measure of relative compound efficacy. For example, a compound will be defined as having a high relative efficacy over another compound if, on the basis of its EC 50 values, it evokes effects in (i) but not in (ii) at a lower relative concentration.
  • identification of agents that preferentially activate the high affinity CGRP receptor includes the step of detecting differential binding to the high affinity CGRP receptor (e.g., the skeletal muscle CGRP receptor) and the metabolic amylin receptor (e.g., the skeletal muscle amylin receptor). Binding studies can be performed conveniently through radioligand binding experiments using a number of several approaches.
  • the test agent is radiolabeled with a suitable radioprobe (e.g. 125 I or 3 H) and incubated with preparations (e.g. skeletal muscle membranes) containing the high affinity CGRP receptor in the presence and absence of excess and unlabelled ligand.
  • a suitable radioprobe e.g. 125 I or 3 H
  • a saturation curve is then constructed and the binding parameter, K d (the concentration of test agent required for half-saturation) derived for specific binding.
  • specific binding parameters of the test agent with the high affinity CGRP receptor can be determined through competition displacement of radiolabeled CGRP ligand.
  • radiolabeled CGRP is incubated with incubated with preparations (e.g. skeletal muscle membranes) containing the high affinity CGRP receptor in the presence of increasing amounts of unlabelled test agent.
  • a plot of bound CGRP ligand against the concentration (log) of unlabelled test agent will yield a sigmoidal curve from which a inhibition constant, Ki, can be derived for the test agent.
  • a high affinity CGRP receptor agonist (or a test compound that may be an agonist) is administered to animals, in a screening assay to determine whether the compound is useful for reduction in lipid content in an animal (e.g., animal tissue).
  • the agonist can be administered to a non-human animal that serves as a model for human diseases or conditions associated with muscle fat accumulation.
  • mice Examples of such experimental models include animal models for insulin resistance, e.g., the ob/ob (Kreutter, 1991 Diabetes 40[Suppl 1]:159A (Abstract); Bretherton, Endocrinol 129[Suppl]:91A (Abstract)), the db/db (Kreutter, 1991 Diabetes 40 [Suppl 1]:159A (Abstract)), and obese-diabetic viable yellow (Gill, 1991 Life Sci 48:703-710) strains of mice, and the A/N-cp (Huang, 1992 Hypertension 19[Suppl I]:101-109) (Fig.
  • an EC 50 of an agent (or mixture of test agents, e.g., in some high throughput screening formats) for the high affinity CGRP receptor and/or metabolic amylin receptor is to be determined
  • a variety of assays can be used.
  • an EC 50 for a specified compound, receptor and effect can be determined by assaying the effect of various concentrations of the ligand on binding of CGRP-1 to the high affinity CGRP receptor and generating a dose-response curve of agent concentration versus fatty acid release.
  • various concentrations of the test agent are incubated with isolated soleus muscle strips under conditions that maintain tissue viability.
  • triglyceride and free fatty acids are extracted, quantitated, and plotted against the logarithm of the test agent concentration.
  • a sigmoidal curve is fitted to the data and a EC 50 value derived from the concentration of test agent required for 50% maximal stimulation.
  • An exemplary assay and resulting dose- response data are described in the examples, infra. See Example 4.
  • a range of concentrations of CGRP can be added to a sample containing a population of the receptors (e.g., skeletal muscle). By plotting CGRP concentration vs.
  • the EC 50 for CGRP binding can be determined as the CGRP concentration that causes half-maximal stimulation of the effect being determined, e.g., free fatty acid production.
  • an agent (or plurality of agents, for example, in pools or in a high throughput screening format as described infra) is (a) assayed to determine the EC 50 of the agent for stimulating lipolysis via the high affinity CGRP receptor (e.g., the skeletal muscle CGRP receptor), i.e., U EC 50-CGRP - R " and (b) assayed to determine the EC 50 for stimulating lipolysis, i.e., "ECso- am yii n -ii"
  • the two EC 50 's are compared and agents with a EC5 0- amyiin- R EC 5 o-cGRP- R ratio greater than about 10 are considered to have a significantly lower EC 50 for the high affinity receptor than the amylin receptor.
  • the ECso-amyii n - R / ⁇ Cso-cGR P - R ratio is at least about 10, often at least about more than about 10 , more than 5 10 , more than about 10 3 , more than 5 x 10 3 , more than about 10 4 , more than about 10 5 , more c n Q than about 10 , more than about 10 , or more than about 10 .
  • Screening can be carried out using a variety of types of compounds and compositions. Suitable test agents include naturally occurring and synthetic compounds or compositions.
  • test agents include polypeptides (including proteins and short peptides, such as the CGRP-related peptides described herein), carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; lipids or phospholipids; fatty acids; steroids; dipeptides, amino acid analogs, organic molecules, e.g., small molecules (e.g., MW less than 1000).
  • the test compounds can be of a variety of chemical types including, but not limited to, heterocyclic compounds, carbocyclic compounds, -lactams, polycarbamates.
  • the agent is identified by screening libraries of compounds.
  • a large number of potentially useful activity-modifying compounds can be screened in extracts from natural products as a source material. Sources of such extracts can be from a large number of species of fungi, actinomyces, algae, insects, protozoa, plants, and bacteria. Those extracts showing activity can then be analyzed to isolate the active molecule. See for example, Turner, 1996, J. Ethnopharmacol 51 :3943; Suh, 1995, Anticancer Res. 15:233-39. Many other test agents and libraries are known in the art. In many embodiments, a library of test agents contains at least 100 different test compounds or agents.
  • the invention provides a method for determining whether an agent is useful for stimulating lipolysis in a mammal by comparing the lipolytic activity of the agent with the lipolytic activity of CGRP.
  • agents that stimulate lipolysis in a cell or tissue (e.g., skeletal muscle or liver) as well or better than CGRP (when compared on an equal molar basis) are considered useful for stimulating lipolysis in a mammal and are candidate compounds for animal or human therapeutics.
  • the step of comparing may be carried out in parallel (done at the same time) assays.
  • the lipolytic activity (e.g., FFA produced per mole agent per unit time under specified conditions) is compared to a standard value previously recorded (e.g., in a computer readable medium or otherwise).
  • the comparison may also be indirect:
  • the lipolytic activity of compound A may be compared to CGRP, and the lipolytic activity of the test agent may be compared with that of compound A (and thus compared indirectly with CGRP).
  • the lipolytic activity of CGRP as mediated by the high affinity receptor is used as the basis for comparison.
  • the comparison is to the lipolytic activity of CGRP at a concentration less than 300 pM.
  • the comparison is to the lipolytic activity of CGRP at a concentration that does not substantially stimulate lipolysis of the metabolic amylin receptor.
  • the lipolytic activity of CGRP as mediated by both the high affinity receptor and the metabolic receptor, or by the metabolic receptor alone is used as the basis for comparison.
  • any form of CGRP that stimulated lipolysis in skeletal muscle may be used; in a related embodiment (as will be apparent from the foregoing) the form of CGRP is one that stimulates lipolysis via the high affinity receptor.
  • Exemplary forms of CGRP are rat and human CGRP-1.
  • the invention provides a method of inhibiting lipolysis in skeletal muscle by administering an antagonist of the high affinity and/or metabolic receptor(s).
  • Inhibition of lipolysis in tissues is useful in both experimental and clinical settings.
  • chronic administration of antagonists may induce compensatory metabolic processes in muscle and liver that lead to increased lipid utilisation (e.g. upregulation of b -oxidation) and result in a desirable reduction in muscle lipid.
  • Agents that inhibit lipolysis are also used in drug screening assays (e.g., as positive and negative controls for identification of lipolysis modulators).
  • the antagonist may be any compound that inhibits lipolysis stimulated by an agent such as CGRP or amylin.
  • the antagonist may inhibit binding or compete for binding to the high affinity CGRP receptor or the lower affinity metabolic amylin receptor (see, e.g., Example 4 infra).
  • the antagonist may be a naturally-occurring compound, such as a protein, peptide, polynucleotide, or small effector molecule, or may be synthetically produced.
  • the antagonists is 8,37 amylin or 8,37 CGRP.
  • 8 ' 37 CGRP and 8 ' 37 amylin refer to variants of CGRP and amylin, respectively, in which the C-terminal first seven amino acids have been removed.
  • Non-peptide CGRP antagonists may also be used. Examples include BIBN4096BS (see, Wu et al, 2002, Biochem Soc. Trans. 30:468-73), “compound- 1” and “compound-2” (see Mallee et al., 2002, JBiol Chem. 277:4294-98).
  • the antagonist may be administered in a therapeutically effective amount to a mammal in need of lipolysis inhibition.
  • the receptor antagonists can be combined with a pharmaceutically acceptable excipient and administered to an animal. Modes of administration are similar to those described, supra, for administration of a receptor agonist.
  • Teflon columns (16 ml), teflon frits and Vac Elut apparatus were from Alltech.
  • Aminopropyl bonded silica 40 ⁇ (Bond Elut) column packing was from Phenomenex; hexane and diethyl ether were from Labscan.
  • Heptane was from Waters Associates Inc and boron trifluoride-methanol complex (BF3 in methanol) was from Aldrich Chemical Company Inc. Butylated hydroxy toluene and all fatty acid methyl ester standards were purchased from Sigma.
  • GLC column DB 225 (30m x 25 mm I.D.) was obtained from J & W Scientific.
  • the standard diet contained 5% fat (w/w), which comprised mainly beef tallow.
  • the triglyceride, total and free fatty acid contents of representative samples of this diet were analyzed, and are shown in Table 3. Rats were anaesthetized by intraperitoneal injection of sodium pentobarbital (45 mg/kg body weight) then sacrificed by cervical dislocation.
  • Free fatty acid and total lipid samples were analyzed by gas chromatography, confirmation of the identity of individual fatty acids by mass spectroscopy, to determine free fatty acid and total fatty acid composition of standard rat chow. Values are meat S.E.M. (n ⁇ 7 at each point)
  • Free fatty acid composition of lard, corn oil and olive oil Values are % of total.
  • Soleus muscles of both legs were excised keeping the tissue immersed in KHB; muscles were separated into two equal parts and transferred to the incubation flasks. At the completion of incubation the muscles were snap frozen in liquid nitrogen, and then trimmed of connective tissue and all visible fat. The muscle strips were stored at -80°C until analysis.
  • Aminopropyl phase (250 mg) was packed into 16 ml teflon columns with teflon frits placed at the top and bottom of the bonded phase. Columns were placed in a Vac Elut apparatus and washed twice with 2 ml portions of hexane (Prasad et al, 1988, J Chromato 428: 221-228; Kaluzny et al, 1985, J Lipid Res 26: 135-140). The dry lipid samples were taken up in two 0.150 ml portions of chloroform and applied to the column under atmospheric pressure.
  • the temperature program consisted of a linear increase from an initial temperature of 80°C to a final temperature of 210°C at a rate of 3°C/rnin followed by a ten-minute period at the final temperature.
  • the quantitation of tissue fatty acids was based on retention times of fatty acid methyl ester standards and relative theoretical response factors.
  • Free fatty acids were assigned based on standards and on GC/MS chromatograms. [0159] Determination of tissue free fatty acids and triacylglycerol concentration by enzymatic analyses. Free fatty acids were separated from total lipid, evaporated under a stream of N 2 and stored at -80°C until analysis. Samples were dissolved in 50 ⁇ l of warm ethanol (35-40 °C), and 0.625 ml of a Triton X-100 (6% w/v) solution was added once the ethanol reached room temperature. The solution was stirred for 30 min, then made up to 0.825 ml with the Triton solution.
  • Free fatty acids were quantified using a commercial method (Boehringer Mannheim) adapted for micro analyses (Cobas Mira, Roche Diagnostics), with palmitic acid standards. Triacyl glycerol levels were quantified in the total lipid fraction (Pointe Scientific) with a glycerol standard.
  • Enzymatic analysis of tissue glycerol Frozen muscle samples were powdered and weighted into 2-ml eppendorf tubes. 0.5 ml of 10% (w/v) trichloroacetic acid was added to each sample, which was then homogenised for 4 minutes in ice. The homogenate was centrifuged and vortexed several times, rested on ice for 2 minutes then centrifuged at 10,000 rpm for 5 min.
  • This example describes the stimulation evoked by various agents on the levels of lipid metabolites in isolated rat soleus muscle tissue. Muscle tissue was treated with amylin, CGRP, norepinephrine, or insulin and the free fatty acids, glycerol, and triglyceride levels were compared with control levels. [0163] The effects of amylin, CGRP, insulin and norepmephrine on free fatty acid content in isolated soleus muscles are shown in Figure 2A. There were significant differences in the free fatty acid content of soleus muscles treated with amylin, CGRP and norepinephrine compared to those in control muscle.
  • Amylin, CGRP and norepinephrine evoked decreases in the intramuscular glycerol content as seen in Figure IB.
  • the glycerol content was (0.03 ⁇ 0.004 nmol/g vs. 0.07 ⁇ 0.007, P ⁇ 0.01), with CGRP (0.04 ⁇ 0.008 nmol/g vs. 0.07 + 0.007, P ⁇ 0.01) and after norepinephrine treatment (0.04 ⁇ 0.005 nmol/g vs. 0.07 ⁇ 0.007, P ⁇ 0.01).
  • FIG. 2C shows triacylglycerol content in soleus muscles after 60-min incubation.
  • Treatment with norepinephrine lowered the triglyceride content (0.81 ⁇ 0.09 ⁇ mol/g vs. 1.17 ⁇ 0.15).
  • There was also a tendency toward elevated muscle triglyceride with amylin and CGRP treatment (1.7 ⁇ 0.26 ⁇ mol/g (amylin), 1.9 ⁇ 0.23 (CGRP) vs. 1.17 ⁇ 0.15 (control)), but none of these effects were significantly different from with control values.
  • Table 6 shows the hormone-evoked release of individual free faty acids. This shows that C16:0 is the most abundant free fatty acid present in the soleus muscle, however, incubation in the presence of hormone did not cause an increase in concentration.
  • the intramuscular concentration of C 16:1 (n-9) was decreased on treatment with amylin (0.013 ⁇ 0.0011 nmol/g vs. 0.008 ⁇ 0.0013, P ⁇ 0.05), CGRP (0.013 ⁇ 0.0011 nmol/g vs. 0.008 ⁇ 0.0006, P ⁇ 0.01) and norepinephrine (0.013 ⁇ 0.001 lnmol/g vs. 0.007 ⁇ 0.0007, P ⁇ 0.01).
  • Cervonic acid concentration was also increased on treatment with all three hormones: amylin (0.01 ⁇ 0.0008 nmol/g vs. 0.03 ⁇ 0.004, P ⁇ 0.01), CGRP (0.01 ⁇ 0.0008 nmol/g vs. 0.022 ⁇ 0.002, P ⁇ 0.01) and norepinephrine (0.01 ⁇ 0.0008 nmol/g vs. 0.022 ⁇ 0.0015, P ⁇ 0.05).
  • Hormone evoked release of individual free fatty acids, over 1 hour, in soleus muscle was analyzed by gas chromatography, w corrfirmation the of identity of individual free fatty acids by mass spectroscopy. Values are nmol/g wwt and are expressed as means S.E.M. (n 16 for each point). * Significant difference compared with control (P ⁇ 0.05). ** Significant difference compared w control (P ⁇ 0.01).
  • Table 7 shows the percentage of intramuscular free fatty acid compared to total fatty acid levels. This Table also shows the change in the percentage of the free fatty acids available after hormone treatment.
  • High fat fed rats are a model for insulin resistance, and are a especially useful model (compared to normal-fed animals) in the study of type 2 diabetes mellitus.
  • Table 4, supra shows the composition of the fats used to create the high fat diets.
  • Tables 8, 9, & 10 show the individual free fatty acid content of soleus muscles from rats fed one of the three high fat diets.
  • Figure 16 shows that rats fed a high fat diet are insulin resistant, as determined by insulin response of the soleus muscle. Insulin dose response curves were measured in soleus muscle from rats fed a normal diet ( ⁇ ) or a high fat diet ( ⁇ ) for 51 days. Insulin responses were measured through incorporation of D[ 14 C(U)] glucose into muscle glycogen following incubation for 2 h with various concentrations of insulin.
  • Muscle glycogen was then extracted and analyzed for D[ 14 C(U)] glucose content by liquid scintillation spectrometry.
  • Figure 6 shows the effects of the three hormones on the triglyceride content in soleus muscle of rats fed one of three high fat diets.
  • Figure 6A In the group fed a diet containing 40% added lard (Figure 6A), all three hormones caused significant decrements in soleus muscle triglyceride content compared to the corresponding control (0.75 ⁇ 0.08 (CGRP), 0.71 ⁇ 0.07 (amylin) and 0.69 + 0.09 (norepi) ⁇ mol/g vs. 1.14 ⁇ 0.16 ⁇ mol/g, P ⁇ 0.05 in each case).
  • Figure 7 shows the effects of the three hormones on the free fatty acid content in the soleus muscle of rats fed one of three high fat diets.
  • FIG. 17A shows CGRP-1 evoked a dose-dependent increase in muscle NEFA content with an EC 50 similar to the high affinity site observed in muscle from normal fed animals (0.7 pM + 0.4 to 1.0; mean ⁇ 95% CL).
  • Figure 17B shows that, associated with the increase in NEFA content was a dose-dependent decrease in total intramuscular triglyceride content, an effect not observed in soleus from normal- fed animals.
  • the EC 50 for the decrease in intramuscular triglyceride (0.25 pM ⁇ 0.03 to 1.96; mean ⁇ 95% C.I.) was identical within experimental error to the observed EC 50 for the increase in NEFA content.
  • Basal triglyceride content in muscle from high fat-fed animals was significantly elevated compared to muscle from normal-fed animals.
  • Hie animals were fed a diet high in saturated fat (lard) for 30 days from the time of weaning.
  • Hormone evoked release of individual free fatty acids, over 1 hour, in soleus muscle of rats fed a diet high in corn oil was analyzed gas chromatography, with confirmation the of identity of individual free fatty acids by mass spectroscopy. Values are nmolg wwt are expressed as means ⁇ S.E.M. (n 16 for each point). * Significant difference compared with control (P ⁇ 0.05). ** Signific difference compared with control (P ⁇ 0.01).
  • Hormone evoked release of individual free fatty acids, over 1 hour, in soleus muscle of rats fed a diet high in olive oil was analyzed gas chromatography, with corrfirmation the of identity of individual free fatty acids by mass spectroscopy. Values are nmol/g wwt a are expressed as means ⁇ S.E.M. (n 16 for each point). * Significant difference compared with control (P ⁇ 0.05). ** Signific difference compared with control (P ⁇ 0.01).
  • linear antagomstic peptides both lack the first seven amino acids, and therefore an homologous NH 2 -terminal cys2-cys7 disulfide ring structure shared by the full-length peptides from which they were derived.
  • CGRP CG-terminal cys2-cys7 disulfide ring structure
  • Amylin-(8-37) suppressed the ability of CGRP- 100 nM to increase soleus muscle free fatty acid content compared to incubation with 100 nM CGRP alone (34 ⁇ 5 nmol/g vs. 334 ⁇ 52, P ⁇ 0.01), see Figure 3B.
  • the CGRP antagonist, CGRP-(8-37), also reversed the increase of free fatty acids evoked by 100 nM CGRP (90 ⁇ 7 nmol/g vs. 334 ⁇ 52, P ⁇ 0.01).
  • Addition of maximally stimulating insulin suppressed the ability of 100 nM amylin (216 ⁇ 26 nmol/g vs. 100 ⁇ 11, P ⁇ 0.01) or CGRP (334 ⁇ 52 nmol/g vs. 107 ⁇ 17, P ⁇ 0.01) to evoke an increase in the soleus free fatty acid content.
  • insulin antagonizes the effects of both these hormones to elicit release of free fatty acids in skeletal muscle.
  • cross-reactivity of ' CGRP is consistent with the ability of this antagonist to completely reverse amylin-inhibition of insulin-stimulated glycogen synthesis and amylin-induced increases in blood glucose and lactate (Wang et al., 1991, FEBS Lett. 291:195-198).
  • This order of antagonist potency, antagonist order is also consistent with the hypothesis that CGRP and amylin elicit effects on glucose metabolism in skeletal muscle via binding to an endogenous Cl ins- /ramp 1 receptor.
  • the CGRP receptor antagonists reversed the effects of CGRP.
  • the decrease in triglyceride content evoked by either 1 pM or 100 nM CGRPl was inhibited by a 10-fold molar excess of either antagonist.
  • a 100 pM concentration of antagonist was sufficient to ameliorate the effects of 1 pM CGRP on soleus muscle triglyceride and free fatty acid contents.
  • CGRP-2 has a different mechanism of action than its counterpart, is in fact similar to amylin.
  • the EC 50 value is 5.2 nM ⁇ (2.3 to 11.5; mean ⁇ 95% C.I.) with a corresponding r value of 0.78.
  • Increased intramuscular content of free fatty acids evoked by amylin and CGRP allow for significantly greater potential energy compared to control (2.73 ⁇ 0.28 ⁇ mol g vs. 1.97 ⁇ 0.31, P ⁇ 0.01), (2.96 + 0.22 ⁇ mol/g vs. 1.97 ⁇ 0.31, P ⁇ 0.01).
  • Potential metabolic energy available from free fatty acids after norepmephrine treatment was not significantly different to control (2.24 ⁇ 0.17 ⁇ mol/g).
  • This example describes the materials and methods used in the experiments described in Examples 8-14, including preparation of cells expressing a recombinant receptor.
  • Rat amylin whose sequence is identical to that of murine amylin (Cooper et al., 1994, Endocr. Rev. 15:163- 201, 1994), and is hereinafter designated 'amylin' (lot 0538912); rat CGRP-1 ('CGRP'; lot 501460); rat calcitonin (lot ZJ375); salmon calcitonin ('sCT'; lot ZM423); 8,37 rat amylin (lot 515439); and 8,37 rat CGRP-1 (lot 501395) were purchased from Bachem (Switzerland).
  • Human adrenomedullin was synthesised as previously described (Heller et al., 2000, Anal. Biochem. 285:100-104). Integrity and purity of all peptide preparations were confirmed by mass spectrometry (matrix-assisted laser-desorption ionisation tirne- of-flight MS, MALDI-TOF; Hewlett-Packard G2025A; 4-OH-cinnamic acid matrix). Soluble human insulin was employed throughout as the insulin agonist ('insulin'; Actrapid 100U, Novo Nordisk). Bulk synthetic peptides were stored as powders desiccated at -80 °C under argon until use.
  • Reaction conditions were: tris-HCl (50 mM); KC1 (40 mM); MgCl 2 (5 mM); Tween-20 (0.5%); dithiothreitol (10 mM); RNase inhibitor (20 U); ExpandTM reverse transcriptase (50 U) (Boehringer Mannheim); dNTP's (1 mM); pH 8.3.
  • Oligonucleotide primers for ramp 1 and ramp 3 respectively were 5- TTAGGATCCGTTGCCATGGCCCGGCTGCGGCTCCCG-3 (sense)/5-CGAGAAT TCCTCATCACCCGGGATACCTA-3 (antisense) and 5-ATAGGATCCTG CATCTTAGTTGGCCATGA-3 (sense)/5-ATAGAATTCATCCAGCAGATCCTCA AGC-3 (antisense).
  • Underlined nucleotides respectively indicate BamHI and EcoRI restriction sites introduced to facilitate cloning.
  • PCR amplification was performed in an Eppendorf Mastercycler R gradient Thermal cycler for 40 cycles of 94 °C (1 min), 55 °C (2 min), and 72 °C (3 min).
  • Reaction conditions were: Tris/HCl (10 mM), KC1 (50 mM), MgCl 2 (1.1 mM), oligonucleotides (0.5 ⁇ M), gelatin (0.01 % w/v), dNTP's (200 ⁇ M), 2.5 U REDTaqTM DNA polymerase (Sigma, Saint Louis), cDNA template (10 ng), at pH 8.3.
  • PCR products were isolated and subcloned into pcDNA3.1+ (Invitrogen) through the BamHI and EcoRI restriction sites. Sequence analysis confirmed the fidelity of sequences for both ramp 1 and ramp 3.
  • CTCTATTTCTAGACTGACTCCC-3 antisense
  • underlined residues refer to Pstl and Xbal restriction sites introduced to facilitate cloning, while the bolded residue denotes a point mutation that introduced a restriction site.
  • RT-PCR was carried out using ElongaseTM. Amplification was performed at 94 °C (30 s), 56 °C (30 s), 68 °C (2 min) for 5 cycles, then at 94 °C (30 s), 60 °C (30 s), 68 °C (2 min) for 30 cycles.
  • L6 myoblasts and cos-7 cells were obtained from ATCC Both cell lines were cultered in Dulbecco's modified Eagle's medium (DMEM; Gibco/BRL, high glucose) supplemented with 10 % fetal calf serum (Gibco/BRL, Gaithersburg, MD), L-glutamine (2 mM), penicillin (100 U/ml; Sigma), streptomycin (100 mg/ml; Sigma), saline (0.85 %), under 5 % C0 2 /95% air at 37 °C The day before transfection, cells were trypsinised and seeded into six-well plates at a density of 2.9 x 10 5 cells/well in the same medium.
  • DMEM Dulbecco's modified Eagle's medium
  • Gibco/BRL high glucose
  • Binding conditions were selected on the basis of previous time-course experiments (data not shown). Cells were washed twice with this buffer, then incubated with 1 ml of the same plus radioligand for 4 h at room temperature (21 °C). Next, cells were washed twice with ice-cold binding buffer, and cell-bound radioactivity counted by liquid scintillation spectrometry following transfer of cells with two washes of 0.5 M sodium hydroxide. [0193] Northern blot analysis. Total RNA was isolated using TrizolTM Reagent (Life Technologies) according to manufacturer's instructions.
  • RNA 5 ⁇ g/lane was electrophoresed on a formaldehyde/agarose gel (1 % w/v), transferred to a Nylon Hybond TM membrane (Amersham Pharmacia Biotech) then cross-linked with a Stratalinker 2400 (Stratagene, La Jolla, CA).
  • Mouse ramp 1 and ramp 3 cDNA hybridisation probes were random primed with [ ⁇ - PjdCTP. Filters were pre-hybridised for 2 h at 65 °C in Church- Gilbert solution (0.25 M disodium phosphate, 7 % SDS, 1 mM EDTA), then hybridisation performed overnight at 65 °C in the same buffer containing 25 ng/ml of labelled probe.
  • Soleus muscles were dissected under oxygenated KHB (118 mM NaCI, 4.75 mM KC1, 1.2 mM MgS0 4 , 24.8 mM NaHC0 3 , 1.2 mM KH 2 P0 4 , 2.54 mM CaCl 2 , 10 mM glucose, pH 7.4) and teased into halves, then incubated as described below.
  • KHB 118 mM NaCI, 4.75 mM KC1, 1.2 mM MgS0 4 , 24.8 mM NaHC0 3 , 1.2 mM KH 2 P0 4 , 2.54 mM CaCl 2 , 10 mM glucose, pH 7.4
  • cAMP concentration was measured in cells seeded in six-well plates and transfected as described. Following aspiration, cells were incubated for 15 min at room temperature in the presence of stated concentrations of ligands in binding buffer. Isobutylmethylxanthine (Sigma) was added to a final concentration of 0.5 mM together with stated concentrations of peptides, and the reaction terminated by aspiration. Cells were washed with 0.5 ml ice-cold TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7.6) and rapidly transferred to eppendorf tubes in 1 ml of the same buffer, then boiled for 5 min.
  • muscle strips were transferred to ice-cold KHB for 5-10 min to remove excess [ 3 H]DOG and [ 14 C]sorbitol. They were then blotted, frozen, tendons excised, then lyophilized for a further 48 h. Muscles were then weighed, transferred to scintillation vials and digested with 0.5 ml 1 M NaOH at 60 °C for 1 h. Scintillation fluid (4 ml) was added to each vial, and samples counted in a Beckman liquid scintillation spectrometer, preset to count 14 C and H channels simultaneously.
  • the amount of each isotope present in the samples was determined and used to calculate the extracellular space and the intracellular concentration of [ 3 H]DOG.
  • the accumulation of intracellular [ 3 H]DOG, a measure of ⁇ muscle glucose uptake, was calculated by subtracting the concentration of [ H]DOG in the extracellular space from the corresponding total muscle [ 3 H]DOG]; extracellular [ 3 H]DOG was quantified by measuring the muscle concentration of [ 14 C]sorbitol. [0198] Measurement of glycogen metabolism in isolated, incubated soleus muscle.
  • Glycogen was precipitated overnight at -20°C with 96 % ethanol (1.2 ml), followed by centrifugation at 8,000g (15 min, 4 °C). Pellets were washed twice with the same amount of ethanol then dissolved in water (0.6 ml). Aliquots (0.3 ml) were analyzed for D[ 14 C(U)] glucose content by liquid scintillation spectrometry.
  • Total glycogen content was measured in muscle strips by lyopbilization followed by hydrolysis of glycogen to D-glucose with amyloglucosidase (Sigma) 20 U/100 ml in 0.2 M sodium acetate/acetic acid buffer, pH 4.8, and expressed as ⁇ mol 'glucosyl' units/g dry muscle weight.
  • Free glucose in supernatant solutions was measured with a glucose/lactate analyzer (YSI 2300STAT, Yellow Springs Corporation) using D- glucose oxidase immobilized enzyme chemistry.
  • K ⁇ values were calculated by the method of Cheng and Prusof (Cheng et al., 1973, Biochem. Pharmacol. 22:3099-3108) using a radioligand concentration of 20 nM and a K d of 9.2 that was derived independently from a saturation curve. 95% confidence intervals (nM) are bracketed.
  • Amylin evoked a maximal increase in cAMP content of about 2-fold when added at either 10 nM or 100 nM (Figure 13B); the magnitude of this effect was not different in the absence or presence of insulin (23.7 nM). Picomolar concentrations of amylin had no effect on muscle cAMP levels, illustrating a difference in the activity of amylin and CGRP-1 ( Figure 13C).
  • Rat soleus muscle strips were incubated with amylin, CGRP-1 or sCT in the absence or presence of a maximally effective concentration of human insulin (23.7 nM), as shown in Fig. 15. Significance was determined by two-way ANOVA.
  • Body temperature was maintained at 37°C throughout surgery and the experiment by a heating pad.
  • the carotid artery and jugular vein were cannulated with a solid-state blood pressure transducer and a saline filled PE 50 catheter respectively.
  • surgery was similar to above with the alteration being that both the carotid artery and jugular vein were cannulated with saline filled PE 50 catheters.
  • Fluids saline or hormone dissolved in saline
  • the carotid artery line was connected to a blood pressure transducer. Blood samples were taken from this line at time zero (baseline reading), 30 minutes and every 20 minutes until the end of the experiment at 90 minutes.
  • Infusion was a combination of antagonist, CGRP or saline. Animals received either antagonist or saline for the first 30 minutes at a rate of lml/kg/h, the second line was then started, containing either CGRP or saline as appropriate, again at a rate of lml/kg/h.
  • MAP mean arterial pressure
  • HR heart rate
  • HR heart rate
  • Oximeter Nonin 8600V Pulse Oximeter
  • core body temperature was all continuously monitored throughout the experiment using a PowerLab/16s data acquisition module. Calibrated signals were displayed on screen and saved to disc as 2 s averages of each variable.
  • Statistical significance was analysed by oneway ANOVA followed by post-hoc analysis by Dunnetts Multiple Comparsion Test. Significant difference compared to control: * P ⁇ 0.05; **P ⁇ 0.01; *** P ⁇ 0.001.
  • the experiments in Examples 15 and 16 show that a dose of CGRP reached at infusion of 100 pmoles/kg/hr is sufficient to evoke effects on lactate and glucose production, lipid metabolism, and vasodilatation.
  • a CGRP-1 serum concentration of 300 pM was observed in the 100 pmoles/kg/hr infused animals, representing a steady state concentration over the hour infusion period.
  • Masoprocol (nordihydroguaiaretic acid), is a lipoxygenase inhibitor, that has been identified as the active component of Creosote bush extracts. Oral administration of this compound lowers serum NEFA and triglyceride concentrations in rats with fructose- induced hypertriglyceridemia (Gowri et al. 2000, Am. J. Physiol. 279: E593-E600), and decrease blood glucose and triglyceride concentrations in a fat-fed/streptozotocin rat model (Reed et al. 1999, Diabetologia 42: 102-106).
  • masoprocol has been shown to exert its antilipolytic actions in adipocytes via inhibition of hormone sensitive lipase (Gowri et al. 2000, Am. J. Physiol. 279: E593-E600), its Effects on CGRPl-evoked effects on NEFA and triglyceride contents in soleus muscle from high fat-fed rats were investigated.

Abstract

L'invention concerne des procédés permettant de stimuler la lipolyse dans des cellules et tissus mammaliens par contact de la cellule ou du tissu avec un agoniste d'un récepteur de CGRP, par exemple le récepteur de CGRP à affinité élevée. L'agoniste peut, de préférence, stimuler le récepteur CGRP à affinité élevée en comparaison avec le récepteur d'amyline métabolique. L'invention concerne également des procédés de criblage permettant d'identifier des agonistes de récepteur, des compositions renfermant de tels agonistes et des schémas thérapeutiques faisant appel à de tels agonistes.
EP02803945A 2001-11-26 2002-11-26 Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens Withdrawn EP1461068A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US33342201P 2001-11-26 2001-11-26
US333422P 2001-11-26
NZ51573101 2001-11-26
NZ51573101 2001-11-26
PCT/NZ2002/000262 WO2003045424A1 (fr) 2001-11-26 2002-11-26 Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens

Publications (2)

Publication Number Publication Date
EP1461068A1 true EP1461068A1 (fr) 2004-09-29
EP1461068A4 EP1461068A4 (fr) 2006-03-29

Family

ID=26652295

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02803945A Withdrawn EP1461068A4 (fr) 2001-11-26 2002-11-26 Procedes et compositions pour normaliser les niveaux lipidiques dans les tissus mammaliens

Country Status (6)

Country Link
EP (1) EP1461068A4 (fr)
JP (1) JP2005523418A (fr)
CN (1) CN1617737A (fr)
AU (2) AU2002356469A1 (fr)
CA (1) CA2471833A1 (fr)
WO (1) WO2003045424A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1115389B1 (fr) 1998-09-25 2014-03-12 PhilERA New Zealand Limited Fructosamine-oxydase: antagonistes et inhibiteurs
ATE555782T1 (de) 2002-03-08 2012-05-15 Philera New Zealand Ltd Prävention und/oder behandlung von kardiovaskulären erkrankungen und/oder damit zusammenhängender herzinsuffizienz
CA2496411A1 (fr) 2002-08-20 2004-03-04 Protemix Corporation Limited Formes posologiques et traitements s'y rapportant
DK1778618T3 (da) 2004-07-19 2014-03-10 Philera New Zealand Ltd Syntese af triethylentetraminer
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates
SI3495392T1 (sl) 2011-05-20 2021-11-30 H. Lundbeck A/S Sestavki proti CGRP in uporaba le-teh
AR086515A1 (es) 2011-05-20 2013-12-18 Alderbio Holdings Llc Uso de anticuerpos o fragmentos de anticuerpos anti-cgrp o anti-cgrp-r para tratar o prevenir las formas cronicas y agudas de la diarrea
CN107827982B (zh) 2011-05-20 2021-07-06 H.伦德贝克公司 抗cgrp抗体和抗体片段用于在有需要的受试者中预防或抑制畏光或厌光的用途
CA2916980C (fr) * 2013-07-03 2023-02-21 Alder Biopharmaceuticals, Inc. Regulation de metabolisme de glucose a l'aide d'anticorps anti-cgrp
AU2020206241A1 (en) 2019-01-08 2021-08-26 H. Lundbeck A/S Acute treatment and rapid treatment of headache using anti-CGRP antibodies

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0309100A2 (fr) * 1987-08-26 1989-03-29 Amylin Pharmaceuticals, Inc. Emploi de l'amyline ou CGRP pour le traitement du diabète sucré
WO1996004928A1 (fr) * 1994-08-16 1996-02-22 Human Genome Sciences, Inc. Recepteur peptidique apparente genetiquement a la calcitonine
EP0845269A2 (fr) * 1996-11-29 1998-06-03 Weihan Wo Composition lyposomale contenant le peptide apparenté au gène de calcitonine et sa préparation

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5175145A (en) * 1988-08-26 1992-12-29 Amylin Pharmaceuticals, Inc. Treatment of diabetes mellitus with amylin agonists
AU6524794A (en) * 1993-03-24 1994-10-11 Amylin Pharmaceuticals, Inc. Cloned receptors and methods for screening
WO1998003534A1 (fr) * 1996-07-23 1998-01-29 Smithkline Beecham Corporation Facteur composant du recepteur du peptide apparente au gene de la calcitonine (houdc44)
JP4372345B2 (ja) * 1998-02-13 2009-11-25 アミリン・ファーマシューティカルズ，インコーポレイテッド 新規な混合アミリン活性化合物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0309100A2 (fr) * 1987-08-26 1989-03-29 Amylin Pharmaceuticals, Inc. Emploi de l'amyline ou CGRP pour le traitement du diabète sucré
WO1996004928A1 (fr) * 1994-08-16 1996-02-22 Human Genome Sciences, Inc. Recepteur peptidique apparente genetiquement a la calcitonine
EP0845269A2 (fr) * 1996-11-29 1998-06-03 Weihan Wo Composition lyposomale contenant le peptide apparenté au gène de calcitonine et sa préparation

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHATZIPANTELI K ET AL: "CALCITONIN GENE-RELATED PEPTIDE IS AN ADIPOSE-TISSUE NEUROPEPTIDE WITH LIPOLYTIC ACTIONS" ENDOCRINOLOGY AND METABOLISM, LONDON, GB, vol. 3, no. 4, 1996, pages 235-242, XP008057490 ISSN: 1074-939X *
HETTIARACHCHI M ET AL: "Rat amylin-(8-37) enhances insulin action and alters lipid metabolism in normal and insulin-resistant rats" AMERICAN JOURNAL OF PHYSIOLOGY, vol. 273, no. 5 PART 1, November 1997 (1997-11), pages E859-E867, XP002362126 ISSN: 0002-9513 *
MOORE MARY COURTNEY ET AL: "Insulin- and glucagon-independent effects of calcitonin gene-related peptide in the conscious dog" METABOLISM CLINICAL AND EXPERIMENTAL, vol. 48, no. 5, May 1999 (1999-05), pages 603-610, XP002362125 ISSN: 0026-0495 *
See also references of WO03045424A1 *
YE J-M ET AL: "EVIDENCE THAT AMYLIN STIMULATES LIPOLYSIS IN VIVO: A POSSIBLE MEDIATOR OF INDUCED INSULIN RESISTANCE" AMERICAN JOURNAL OF PHYSIOLOGY, AMERICAN PHYSIOLOGICAL SOCIETY, BETHESDA, MD, US, vol. 280, no. 4, PART 1, April 2001 (2001-04), pages E562-E569, XP009009879 ISSN: 0002-9513 *

Also Published As

Publication number Publication date
AU2009201147A1 (en) 2009-04-23
CA2471833A1 (fr) 2003-06-05
AU2002356469A1 (en) 2003-06-10
WO2003045424A1 (fr) 2003-06-05
AU2002356469A2 (en) 2003-06-10
CN1617737A (zh) 2005-05-18
JP2005523418A (ja) 2005-08-04
EP1461068A4 (fr) 2006-03-29

Similar Documents

Publication Publication Date Title
AU2009201147A1 (en) Methods and Compositions for Normalizing Lipid Levels in Mammalian Tissues
Guillet et al. Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans
Dyck et al. Insulin increases FA uptake and esterification but reduces lipid utilization in isolated contracting muscle
Winder Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle
Liu et al. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81
US7803557B2 (en) Methods for identification of eicosapentaenoic acid analogs using anti-inflammatory receptors
Canatan et al. Relationship among levels of leptin and zinc, copper, and zinc/copper ratio in plasma of patients with essential hypertension and healthy normotensive subjects
McKie et al. Biochemical adaptations in white adipose tissue following aerobic exercise: from mitochondrial biogenesis to browning
Nakahara et al. Comparison of feeding suppression by the anorexigenic hormones neuromedin U and neuromedin S in rats
Bhat et al. AdipoRon: a novel insulin sensitizer in various complications and the underlying mechanisms: a review
US20040038861A1 (en) Methods and compositions for normalizing lipid levels in mammalian tissues
Tollet-Egnell et al. Growth hormone-mediated alteration of fuel metabolism in the aged rat as determined from transcript profiles
Patte et al. GABA inhibits endozepine release from cultured rat astrocytes
KR20080044360A (ko) Ｃｃｒ2 매개 질병 또는 장애의 치료방법
Hastings et al. Influence of leptin on neurotransmitter overflow from the rat brain in vitro
JP2004533225A (ja) インスリン及びそのアンタゴニストのためのリンタンパク質標的
US20050250215A1 (en) Measurement of melanocortin peptides and uses thereof
US7662780B2 (en) Administering hypocretin to obese individuals
NZ542281A (en) Methods and compositions for normalizing muscle lipid levels using CGRP-1
Tvarijonaviciute et al. Serum adiponectin concentration in dogs—absence of diurnal variation and lack of effect of feeding and methylprednisolone administration
CA2577176A1 (fr) Procede de detection d'agoniste ou d'antagoniste transporteur de carnitine et utilisations
US7947458B1 (en) Placental human neurokinin B precursor
Piao et al. Amylin-induced suppression of ANP secretion through receptors for CGRP1 and salmon calcitonin
Owecki et al. Circulating resisistin levels are lower in hypothyroid women but independent from thyroid hormones concentrations
Nomoto et al. Suppressive Effects of Neuromedin U Receptor 2-Selective Peptide Agonists on Appetite and Prolactin Secretion in Mice

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040625

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1069977

Country of ref document: HK

A4 Supplementary search report drawn up and despatched

Effective date: 20060210

17Q First examination report despatched

Effective date: 20070301

19U Interruption of proceedings before grant

Effective date: 20100713

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: PROTEMIX CORPORATION LIMITED

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1069977

Country of ref document: HK

19W Proceedings resumed before grant after interruption of proceedings

Effective date: 20111004

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: PHILERA NEW ZEALAND LIMITED

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20120204