CN1617737A - Compositions and method for normalizing lipid levels in mammalian tissues - Google Patents
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Abstract
The present invention provides a process for stimulating lipidolysis by contacting cells or tissues with the activation agent of CGRP receptors such as high-affinity CGRP receptor in cells or tissues of mammalian. Relative to metabolism amylopectin receptor, said activation agent preferably stimulates the high-affinity CGRP receptor. The present invention provides a screen method used for determining the receptor activation agent, a pharmaceutical composition containing the activation agent and a therapeutic schedule using the activation agent. The amino acid sequence of the peptides used in experiments are shown as the figure above. The peptide used are rat amylopectin, rat CGRP 1, rat amylopectin-(8-37) and human CGRP-(8-37). A intermolecular disulfide bond exist between the second and the seventh Cys residues for all peptides except the antagonist in which between the ninth and the fourteenth residues.
Description
With reference to related application
The application enjoys in the rights and interests of U.S.'s patent application formerly 60/333,422 of application on November 26 calendar year 2001, and this patent application is hereby incorporated by.
Invention field
The invention provides and be used for regulating compositions and methods and compositions lipid level and that determine to can be used for regulating lipid level at cell or tissue.The present invention can be used for biology and medical domain.
Technical background
Lipid metabolic disorder plays an important role in various diseases and pathological state.For example, accumulation of lipid is important Developmental and Metabolic Disorder in the sufficient evidence explanation skeletal muscle, and expression not only produces insulin resistant at muscle itself but also in liver, lipid.Has are McGarry etc. if Minkowski lost the sense of taste and how about incited somebody to action? is another angle seen diabetes (What if Minkowski had been ageusic? An alternative angle ondiabetes) science, on October 30th, 1992; 258 (5083): 766-70; Ellis etc., long acyl coenzyme A ester is as the indicator (Long-chain acyl-CoA esters as indicators of lipid metabolism and insulinsensitivity in rat and human muscle.) of lipid metabolism in rat and people's the muscle and insulin sensitivity, U.S. physiology endocrine metabolism magazine, in JIUYUE, 2000; 279 (3) E554-60; Furler etc., high lipid meals influence muscle glucose metabolism (A high-fat diet influences insulin-stimulatedposttransport muscle glucose metabolism in rats) after the transhipment of the insulin stimulating in the rat, metabolism, in JIUYUE, 1997; 46 (9): 1101-6; Dobbins etc., the prolongation of muscle carnitine palmityl transferase-1 suppresses to promote rat myocyte inner lipid to be accumulated and insulin resistant (Prolonged inhibition of muscle carnitinepalmitoyltransferase-1 promotes intramyo cellular lipid accumulation andinsulin resistance in rats), diabetes, January calendar year 2001; 50 (1): 123-30; Kraegen etc., behind the liver insulin resistant, in the rat that high lipid is fed, produce muscle insulin resistant (Developmentof muscle insulin resistance after liver insulin resistance in high-fat-fedrats), diabetes, in November, 1991; 40 (11): 1397-403; Unger etc., the lipid toxicity disease (Lipotoxic) of non-lipid tissue in the obesity (Lipotoxic diseases of nonadipose tissuesin obesity), international fat relevant Developmental and Metabolic Disorder magazine (Int J Obes Relat Metab Disord.) in November, 2000; 24 appendix 4:S28-32; Kelley etc., the skeletal muscle fatty acid metabolism relevant, U.S. physiology magazine, in December, 1999 with insulin resistant, obesity and weight loss; 277 (6Pt1): E1130-41); Simoneau etc., can in people's skeletal muscle, utilize the labelling of fatty acid: with insulin resistant and obesity and weight loss result relevant (Markers of capacity to utilize fatty acids in humam skeletalmuscle:relation to insulin resistance and obesity and effect of weightloss), the FASEB magazine, in November, 1999; 13 (14): 2051-60; Manco etc., saturated fatty acid increases directly related (Insulin resistance directlycorrelates with increased saturated fatty acids in skeletal muscletriglycerides) in insulin resistant and the skeletal muscle triglyceride, metabolism, in February, 2000; 49 (2): 220-4.
Insulin resistant is the initial pathogenic process of many human main metabolic diseases, and these diseases have the common basic pathology mechanism that is referred to as " metabolic syndrome " or " X syndrome complex ".The disease that generally is classified as the X syndrome complex comprises 2-type diabetes, hypertension, obesity, dyslipidemia (dyslipidaemia), as Zierath etc., 2000, diabetology (diabetologia) 43:821-35; Bergman etc., 2001, digestion and metabolism magazine 49:119-26; Olefsky etc., 1995, U.S.'s clinical nutriology magazine (Am J ClinNutr.) 61 (4 appendix): 980S-986S; Ginsberg etc., 2000, Journal of Clinical Investigation 106:453-8; Groop etc., 1999, the fat metabolism of diabetes, 1 appendix 1:S1-7; Fujimoto etc., 2000, JAMA 17; 108 appendix 6a:9S-14S; Ferrannini etc., 1986, N Engl J Med., 1986,317:350-7; Landsberg etc., 1999, Clin Exp Hypertens; 21:885-894; Ferrannini etc., 1995, metabolism, 44 (9 appendix 3): 15-7; Kahn etc., 2000, Journal of Clinical Investigation 106:473-81; Ginsberg etc., 2000, cardiovascular danger magazine (J Cardiovasc Risk) 7:325-31; Howard etc., 1999, American Journal of Cardiology (Am J Cardiol) 8; 84 (1A): 28J-32J; Grundy etc., 1999, American Journal of Cardiology (Am J Cardiol) 13; 83 (9B): 25F-29F; Misra etc., cardiovascular danger magazine 7 (3): 215-29).
The expectation exploitation makes the medicine and the therapeutic strategy of the lipid content normalization in the tissue.For example, this medicine and therapeutic strategy can be used for the treatment of insulin resistant.
Summary of the invention
In one aspect, the invention provides a kind of by mammalian tissues (as skeletal muscle or liver) is contacted the method for decomposing with a certain amount of high-affinity CGRP receptor stimulating agent at this tissue stimulation lipid, with respect to metabolism amylopectin receptor, the amount of this agonist preferably stimulates high-affinity CGRP receptor active (as stimulating the activity of high-affinity CGRP receptor, and can not stimulate the activity of metabolism amylopectin receptor) effectively.In one embodiment, agonist is CGRP-1.In one embodiment, tissue and about 10
-15M to 10
-10CGRP-1 contact between the M.In one embodiment, the amount of CGRP-1 is less than 300pM.In one embodiment, this tissue exsomatizes.On the other hand, this method comprises the step that detects the stimulation of in the tissue lipid being decomposed.
On the one hand, the invention provides by mammalian cell such as Skeletal Muscle Cell are contacted with CGRP polypeptide or its functional variant biologically, in the method for this cell moderate stimulation lipid decomposition.In one embodiment, the CGRP polypeptide has natural CGRP polypeptide such as human CGRP's sequence.In one embodiment, the CGRP polypeptide is human CGRP-1 polypeptide, perhaps its functional variant biologically.In one embodiment, this polypeptide or variant preferably stimulate high-affinity CGRP receptor.
On the one hand, the invention provides a kind of by contacting, in the method for mammalian bone flesh or the decomposition of liver moderate stimulation lipid with skeletal muscle or liver and CGRP-1 or by its variant of metabolism receptor for stimulating.This method also comprises the variation (for example by measuring the free fatty acid content in mammal muscle, liver, blood or its hetero-organization) of lipid decomposition level in the monitoring mammalian tissues.
On the one hand, the invention provides a kind of therapeutic scheme, comprise (i) give suffer from or susceptible be characterized as the symptom of accumulation of lipid in the skeletal muscle administration CGRP-1 polypeptide, its biologically functional variant or decompose by its variant of metabolism receptor for stimulating and the lipid of (ii) monitoring in the mammal.
On the one hand, the invention provides by using high-affinity CGRP receptor stimulating agent reduces the skeletal muscle lipid level in the mammal of this treatment of needs method, with this understanding, metabolism amylopectin receptor is not stimulated or preferably stimulates high-affinity receptor by this agonist, wherein this mammal has symptom or the disease that is characterized as accumulation of lipid in the tissue.In one embodiment, this mammal is an insulin resistant.In one embodiment, this method also comprises the variation that detects free fatty acid in the mammalian tissues (for example skeletal muscle, liver or blood).In one embodiment, this method also comprises the process of this symptom of monitoring or disease.In one embodiment, using of agonist can not cause blood sugar level rising in mammal.
Related fields the invention provides a kind of by organizing (for example skeletal muscle or liver) to contact the method that reduces the lipid level in the tissue with CGRP polypeptide or its functional variant biologically.In one embodiment, CGRP polypeptide or variant organize the amount of lipid level to be applied to mammal with effective reduction.In one embodiment, this mammal is an insulin resistant, and polypeptide or variant are applied with the amount of insulin resistant in effective reduction mammal.In one embodiment, this polypeptide or variant preferably stimulate high-affinity CGRP receptor.
In one aspect, the invention provides the CGRP polypeptide, be used for reducing the medicament of mammalian bone flesh lipid level or be used for the treatment of purposes in the medicament of insulin resistant in preparation in preparation as CGRP-1 or its functional variant biologically.
On the one hand, the invention provides a kind of method that contacts the lipid decomposition that stimulates in this cell by the cell that will express high-affinity CGRP receptor with high-affinity CGRP receptor stimulating agent.In one embodiment, with respect to metabolism amylopectin receptor, this agonist preferably activates high-affinity CGRP receptor.
In related fields, the invention provides a kind of method by stimulating the lipid in the mammal to decompose for administration high-affinity CGRP receptor stimulating agent.In one embodiment, with respect to metabolism amylopectin receptor, this agonist preferably activates high-affinity CGRP receptor.In one embodiment, this agonist is a polypeptide, as the functional variant on CGRP polypeptide or its biology.In various embodiments, this method comprises measures free content of fatty acid in the mammalian tissues (skeletal muscle, liver, serum, blood plasma or blood), or administration CGRP is given in monitoring influence to the mammal insulin resistant.In one embodiment, agonist decomposes with the effective stimulus lipid and does not cause that vasodilative amount is applied.In related fields, the invention provides a kind of by using the method that a certain amount of high-affinity CGRP receptor stimulating agent stimulates the middle lipid of the mammalian tissues (for example skeletal muscle or liver) that needs this treatment to decompose, with respect to metabolism amylopectin receptor, the content of agonist is enough to preferably activate the amount of high-affinity CGRP receptor.
In various embodiments, the mammal that is applied agonist in skeletal muscle in (for example insulin resistant, X syndrome, 2 one type diabetes) or the liver (for example degeneration of liver lipid or " lipid liver ") have the disease or the symptom of the accumulation of lipid of being characterized as.
In one embodiment of the invention, agonist is applied with pharmaceutically acceptable carrier.The present invention also provides high-affinity CGRP receptor stimulating agent (as the CGRP-1 polypeptide) to be used for the treatment of the purposes of the medicament of the symptom that is characterized as accumulation of lipid in mammalian tissues (for example skeletal muscle or liver) or disease (as insulin resistant or X syndrome) in preparation.In related fields, the invention provides CGRP-1 and be used for the treatment of in preparation and suffer from or susceptible is characterized as purposes in the mammiferous medicament of the symptom of accumulation of lipid in the tissue (for example skeletal muscle or liver), wherein medicament produces the agonist level that is lower than 300pM or be created in about 10 when being administered to mammal in blood in blood when being applied to mammal
-15M to 10
-10Lower agonist level between the M.In one aspect, the invention provides CGRP-1 and be used for the treatment of in preparation and suffer from or susceptible is characterized as purposes in the mammiferous medicament of the symptom of accumulation of lipid in the tissue (for example skeletal muscle or liver), wherein medicament is created in about 10 in blood when being applied to mammal
-15M to 10
-10Agonist level between the M.In one embodiment, this symptom is diabetes, insulin resistant or X syndrome.
The present invention also provides by determining that whether a kind of reagent be that the agonist of CGRP receptor such as high-affinity CGRP receptor determines whether this reagent can be used for stimulating the method for lipid decomposition in the mammal.In one embodiment, this method also comprises definite, preferably activates high-affinity CGRP receptor with respect to this reagent of metabolism amylopectin receptor.In one embodiment, this method also comprises the EC that relatively influences by the reagent of the receptor-mediated reaction of metabolism amylopectin (as the effect to carbohydrate metabolism)
50With the EC of this agents influence by the receptor-mediated reaction of high-affinity CGRP (increasing) as free fatty acid in skeletal muscle tissue or the Skeletal Muscle Cell
50In one embodiment, with the skeletal muscle external test EC that exsomatizes
50Value.In one embodiment, effectively the carbohydrate metabolism influence is the variation of tissue or blood glucose and/or lactate level and/or tissue glycogen's level.
Whether the present invention also provides by determining whether a kind of chemical compound can be used as a kind of method of therapeutic agent, decompose at the mammalian tissues moderate stimulation lipid of expressing high-affinity CGRP receptor and metabolism amylopectin receptor by determining reagent; Whether with respect to metabolism amylopectin receptor preferably stimulate high-affinity CGRP receptor, wherein preferably stimulate the reagent of high-affinity CGRP receptor to be confirmed as useful as therapeutics with definite lipid if decomposing stimulation reagent.This or additive method of the present invention can be used for screening the chemical compound as medicine.In one embodiment, drug screening comprises with different reagent at least 25 (for example side by side or sequentially) and implements preceding method.
In one embodiment, the invention provides a kind of screening technique, comprise that (i) determines a large amount of reagent in conjunction with high-affinity CGRP receptor; (ii) from (i), screen a kind of reagent that preferably stimulates high-affinity CGRP receptor with respect to metabolism amylopectin receptor.In one embodiment, this method comprises that (i) determines a large amount of reagent in conjunction with high-affinity CGRP receptor; (ii) select a kind of reagent from (i), its influence is by the EC of the receptor-mediated reaction of metabolism amylopectin
50Than the EC of this agents influence by the receptor-mediated reaction of high-affinity CGRP
50Higher.In one embodiment, with skeletal muscle (Tathagata is from rat) the external test EC that exsomatizes
50Value.In one embodiment, influence is by the EC of the receptor-mediated reaction of metabolism amylopectin
50Than the EC of this agents influence by the receptor-mediated reaction of high-affinity CGRP
50At least high 10 times.By the receptor-mediated reaction of high-affinity CGRP can be that free fatty acid increases in skeletal muscle tissue or the Skeletal Muscle Cell, is influence to carbohydrate metabolism by the receptor-mediated reaction of metabolism amylopectin.
In related embodiment, the invention provides and determine whether a kind of reagent can be used for the method that stimulates the mammal lipid to decompose, determine in a large number at the reagent of organizing the moderate stimulation lipid to decompose of expressing high-affinity CGRP receptor by (i); (ii) from (i), screen a kind of reagent that preferably stimulates high-affinity CGRP receptor with respect to metabolism amylopectin receptor.In one embodiment, this tissue is skeletal muscle (Tathagata is from rat).In one embodiment, screened library has about 100 kinds of different detectable of surpassing.
The present invention also provides to determine whether a kind of reagent is used for the method for decomposing at mammal moderate stimulation lipid, comprises relatively the lipid degrading activity of this reagent and the lipid degrading activity of CGRP.
The present invention also provides by using a certain amount of CGRP receptor stimulating agent and treats the method for insulin resistant in the mammal, the amount of this agonist effectively activates high-affinity CGRP receptor in the mammalian tissues and does not activate metabolism amylopectin receptor, and wherein this agonist is a kind of reagent that above-mentioned screening technique is determined.In one embodiment, this Therapeutic Method comprises the amount of measuring free fatty acid in the mammalian tissues or the additional step of monitoring insulin resistant in mammal.
In one aspect, the invention provides the dosage of mensuration high-affinity CGRP receptor stimulating agent or the method for prescription, this dosage or prescription decompose and minimize the side effect do not expected in the mammal (for example blood sugar level raises or stimulates vasodilation in the mammal) at mammalian tissues (as skeletal muscle or liver) moderate stimulation lipid, (a) give the CGRP receptor stimulating agent of test various various dose of administration or prescription by (i); (b) measure the influence that various dosage and prescription decompose the lipid of testing in the mammiferous tissue and measure the influence of various dosage to side effect, carry out the dose-response analysis, lipid decomposes and the dose-response data of side effect thereby set up; (ii) determining to improve lipid from the dose-response data decomposes and does not cause side effect or significantly improve that lipid decomposes and Min. increases the dosage of the CGRP receptor agonism agent prescription of side effect.In one embodiment, determine the influence that various dosage or prescription decompose lipid by free fatty acid levels in the mensuration mammalian tissues.
The present invention also provides and contains the reagent for the treatment of effective dose and the compositions of pharmaceutically acceptable excipient, and this reagent preferably stimulates high-affinity CGRP receptor with respect to metabolism amylopectin receptor.In one embodiment, when being applied to mammal, said composition stimulates the high-affinity CGRP receptor in mammal and does not stimulate metabolism amylopectin receptor.In one embodiment, said composition stimulates lipid to decompose and Min. ground stimulation vasodilation.
In one aspect, the invention provides a kind of mammiferous pharmaceutical composition in unit dose form that is administered to, described unit dose comprises a certain amount of high-affinity CGRP receptor stimulating agent and pharmaceutically acceptable excipient, and the content of agonist effectively produces in blood with respect to the metabolism amylopectin preferably stimulates the agonist level of high-affinity CGRP receptor (for example to stimulate high-affinity CGRP receptor active and can not stimulate metabolism amylopectin receptor active.Agonist is CGRP-1 in one embodiment).In one embodiment, this agonist is CGRP-1 and its functional variant biologically.In one embodiment, this agonist is that the agonist level is less than 300pM in CGRP-1 and the blood.In one embodiment, this agonist be in CGRP-1 and the blood agonist level about 10
-15M is to about 10
-10Between the M.
The present invention also provides high-affinity CGRP-1 receptor to be used for the treatment of in preparation to suffer from or susceptible is characterized as purposes in the mammiferous medicament of symptom of tissue (as skeletal muscle or liver) accumulation of lipid, when being applied to mammal, this medicament produces the agonist of certain level in mammal, being enough to preferably stimulates high-affinity CGRP receptor (for example to stimulate high-affinity CGRP receptor active and can not stimulate metabolism amylopectin receptor active with respect to the metabolism amylopectin.Agonist is CGRP-1 in one embodiment).
On the other hand, the invention provides metabolism amylopectin receptor by effectively suppressing the amount that lipid decomposes to administration and/or high-affinity CGRP metabolism amylopectin receptor (as
8,37Amylopectin or
8,37CGRP) antagonist suppresses the method that lipid decomposes in the mammal.
In yet another aspect, the invention provides by the mammalian tissues (as skeletal muscle) and the agonist (as CGRP) of metabolism amylopectin receptor being stimulated the method for lipid decomposition in the tissue.
Brief description
The aminoacid sequence of used peptide in the accompanying drawing 1 expression test.Used peptide is AC128, rat CGRP-1 (CGRP), AC128-(8-37) and human CGRP-(8-37).Used peptide has intramolecular disulfide bond between the 2nd and the 7th Cys residue." the NH of carboxyl terminal
2" represent this hormone amidatioon.
Accompanying drawing 2A represents the influence to the muscle free fatty acid content of amylopectin, CGRP, norepinephrine and insulin.Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM amylopectin or CGRP-1,4.4 μ M norepinephrine or 23.7nM insulin.Numerical value is average ± S.E.M (respectively organizing n=8).* compare significant difference (p＜0.05) with matched group.* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 2B represents the influence to the muscle glycerol content of amylopectin, CGRP and norepinephrine.Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM amylopectin or CGRP-1,4.4 μ M norepinephrine.Numerical value is average ± S.E.M (respectively organizing n=8).* compare significant difference (p＜0.05) with matched group.* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 2C represents that amylopectin, CGRP and norepinephrine do not have the test of influence to the muscle content of triglyceride.Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM amylopectin or CGRP-1 or 4.4 μ M norepinephrine.Numerical value is average ± S.E.M (respectively organizing n=8).
Accompanying drawing 3A represents that amylopectin-(8-37), CGRP-(8-37) and insulin increase the influence of intramuscular free fatty effect to amylopectin.Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM amylopectin, the 100nM amylopectin with 10 μ M amylopectins-(8-37) (A/A) or human CGRP-(8-37) (A/C), 10 μ M amylopectins-(8-37) (A-8-37) or 100nM amylopectin+23.7nM insulin (A+I).Numerical value is average ± S.E.M (each point n=7).* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 3B represents that amylopectin-(8-37), CGRP-(8-37) and insulin increase the influence of intramuscular free fatty effect to CGRP-1.Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM CGRP, the 100nM CGRP with 10 μ M human CGRPs-(8-37) (C/C) or amylopectin-(8-37) (C/A), 10 μ M human CGRPs-(8-37) (C-8-37) or 100nM CGRP+23.7nM insulin (C+I).Numerical value is average ± S.E.M (each point n=7).* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 3C represents that amylopectin-(8-37) and CGRP-(8-37) increase the influence of intramuscular free fatty effect to CGRP-1.Musculus soleus is cultivated 1h in KHB buffer (contrast), 1pM CGRP, 1pM CGRP+100pM CGRP-(8-37) or 1pM CGRP+100pM amylopectin-(8-37).Numerical value is average ± S.E.M (each point n=7).* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 4A and 4B represent that CGRP-1 is to stimulating the dose-dependent influence of the free fatty acid content in the musculus soleus of being cultivated.Accompanying drawing 4C represents that CGRP-2 is to stimulating the influence of the free fatty acid content in the rat soleus muscle of being cultivated.Musculus soleus is cultivated in KHB buffer (contrast) or certain limit CGRP concentration.Numerical value is average ± S.E.M (each point n=7).
Accompanying drawing 5A represents that amylopectin is to stimulating the dose-dependent influence of the free fatty acid content in the rat soleus muscle of being cultivated.Musculus soleus is cultivated in KHB buffer (contrast) or certain limit amylopectin concentration.Numerical value is average ± S.E.M (each point n=7).
When accompanying drawing 5B represented to exist 100pM CGRP, amylopectin was to stimulating the dose-dependent influence of free fatty acid content.Musculus soleus is cultivated in KHB buffer (contrast) or certain limit amylopectin concentration+100pM CGRP.Numerical value is average ± S.E.M (each point n=7).
Accompanying drawing 6A, 6B and 6C represent the influence to content of triglyceride in the musculus soleus of the rat of the high lipid food of feeding of amylopectin, CGRP and norepinephrine.The food of feeding and forming to rat by 40% Adeps Sus domestica (6A), Semen Maydis oil (6B) or olive oil (6C).Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM amylopectin or CGRP-1 or 4.4 μ M norepinephrine.Numerical value is average ± S.E.M (each point n=7).* compare significant difference (p＜0.05) with matched group.* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 7A, 7B and 7C represent the influence to free content of fatty acid in the musculus soleus of the rat of the high lipid food of feeding of amylopectin, CGRP and norepinephrine.The food of feeding and forming to rat by 40% Adeps Sus domestica (7A), Semen Maydis oil (7B) or olive oil (7C).Musculus soleus is cultivated 1h in KHB buffer (contrast), 100nM amylopectin or CGRP-1 or 4.4 μ M norepinephrine.Numerical value is average ± S.E.M (each point n=7).* compare significant difference (p＜0.05) with matched group.* compares significant difference (p＜0.01) with matched group.
Accompanying drawing 8 expressions [
3H] AC128 or [
3H] salmon calcitonin see calcimar (sCT) with by the myoblastic total and non-specific binding of the L6 that cultivated, this sarcoplast is with the carrier transient transfection that contains described insert.Sarcoplast is following transfected: A only is a carrier; B contains the carrier of Muridae ramp1; C contains reverse insertion isotype (isoform) C1 of rat calcitonin receptor 1
Ins-Carrier; D is respectively with containing Muridae ramp1 and Muridae C1
Ins-The carrier cotransfection.As shown, when lacking (total (T)) and having the corresponding unmark peptide of (non-specific (NS)) 1 μ M, be that the various radioligands of 10nM combine with concentration.In all cases, can obtain the specificity combination by from corresponding total binding, deducting non-specific binding.Each hurdle represent from repeat at least twice the test three times independently the bonded average of sarcoplast transfection (± S.E.M).*, p＜0.05; * *, p＜0.001; With in pairs (unpaired) t-check determine the significance of difference between total and the corresponding non-specific binding.
Accompanying drawing 9 expressions are from using the rat calcitonin receptor isotype 1 (C1 that contains Muridae ramp1 and oppositely insert
Ins-) the L6 sarcoplast displacement of carrier cotransfection bonded [
3H] AC128; When having the unmarked part of prescribed concentration, Muridae ramp1 and C1
Ins-The sarcoplast usefulness of cotransfection [
3H] amylopectin (20nM) cultivation 3h under 21 ℃.Washed cell then, and determine binding radioactivity by the lipid scinticounting.A is with rat CGRP-1 (◆); AC128 (zero); Salmon calcitonin see calcimar (*); Rat calcitonin (8); Or human adrenal gland medullarin (adrenomedullin) displacement [
3H] amylopectin (:) B, use
8,37CGRP (△); Or
8,37Amylopectin (6) displacement [
3H] amylopectin.Each some expression is from the average ± SEM of 3 myoblastic transfections of independence that repeat at least twice test.
Accompanying drawing 10 expression flush end peptide antagonists
8,37Rat CGRP-1 and
8,37The Muridae calcitonin receptor isotype 1 of AC128 to improving albumen 1, ramp1 and oppositely insert with the Muridae receptor active, C1
Ins-Transient cotransfection and the influence of the cAMP concentration in the L6 sarcoplast cultivated with rat CGRP-1 or AC128.Transfected sarcoplast is cultivated 15min with the A of prescribed concentration, CGRP or B amylopectin.A(◆)CGRP；(△)CGRP+
8，37CGRP(5μM)。B:(zero) amylopectin; (6) amylopectin+
8,37Amylopectin (5 μ M) is cultivated.Washed cell is measured muscle cAMP concentration.Each some expression from the average of 3 independent transfections that repeat at least twice test (± SEM).
Accompanying drawing 11 expressions combine with the specificity of the Cos-7 cell of the vector construction body transient transfection shown in the usefulness, use A, [
3H] rat CGRP-1, CGRP B; [
3H] AC128, amylopectin; And C, [
3H]-sCT, the ratio of total binding represent.Specificity in conjunction with comfortable lacking or the difference between each radioligand (being 20nM under each situation) total binding when having corresponding unmarked part (1 μ M).Each some expression is from the average ± SEM of 3 independent transfections that repeat at least twice test.*, p＜0.01 for only with carrier cells transfected combining relatively.
Accompanying drawing 13 expression CGRP and amylopectin are to the concentration dependent effect of the cAMP in the rat skeletal muscle.With shown in the rat CGRP-1 (13A) or AC128 (13B, 13C) the In vitro culture musculus soleus bar of concentration.When lacking or have insulin human (23.7nM), cultivate.Carry out statistical significance by single factor (one-way) ANOVA and detect, then with Dunnett multiple comparisons check carrying out post-hoc analysis, A, * p＜0.05, and compare * * p＜0.01; And B, * p＜0.05, and compare * * p＜0.01.With compare, the cAMP content of measuring in the musculus soleus of handling with 1 μ M isopropyl noradrenalin among A and the B is respectively 3.5 ± 0.2pmol/mg (p＜0.05) and 3.8 ± 0.2pmol/mg (p＜0.01).
Accompanying drawing 14 expression peptide antagonists are to the influence of the amylopectin mediation inhibition of the glucose transfer of the insulin stimulating in the external rat soleus muscle.Separate rat soleus muscle and also peel off, with amylopectin (10nM) and insulin (23.7nM), and with the A of prescribed concentration,
8,37AC128 or B,
8,37Rat CGRP-1 cultivates, with the increase (2-DOG of 2-deoxy-D-glucose; Nmol/g-dry weight/min) determine that glucose shifts.Each point represent to derive from the independent trials of n=4 average (± S.E.M).Carry out statistical significance by single factor ANOVA and detect, then analyze with Dunnett multiple comparisons check carrying out post-hoc; A, * p＜0.05, compare separately with control value corresponding to [insulin (23.7nM)+amylopectin (10nM)] (no antagonist) * * p＜0.01.
Accompanying drawing 15 expression CGRP and related peptides are to baseline in the rat skeletal muscle and metabolic effect external insulin stimulating.Obtain: at AC128, the amylopectin (zero) of prescribed concentration; Rat CGRP-1, CGRP (σ); Or salmon calcitonin see calcimar, in the isolated rat musculus soleus bar of cultivating among the sCT (E) (A, B), total glycogen content; (C, D) D[
14C (U)] conversion of glucose is the dose-response curve of the ratio of glycogen.Lack (A, C) or have that (B D) cultivates during the insulin human of maximal effective dose (23.7nM).Each some expression is from n=4-5 independent trials (total glycogen) with from n=3-6 (D[
14C (U)] conversion of glucose) average (± S.E.M), and derive from n=15 or 12 independent trialss respectively except baseline value, each is measured and repeats 3 times.
The accompanying drawing 16 expression insulin dose-response curve in the rat soleus muscle of high lipid food of feeding.Insulin dose-response curve is to measure from normal diet of feeding (■) or 51 days rat soleus muscle of high lipid food ().Insulin response is after cultivating 2h with various concentration insulins, passes through D[
14C (U)] conversion of glucose is that muscle glycogen is measured.Extract muscle glycogen then and analyze D[by the liquid scintillation spectrophotometer
14C (U)] glucose content.Dose-response curve and the match of S shape dose-response algorithm.The result represents with average ± SEM (each point n=12-18).Significant difference (p＜10 between two curves of total sum of squares analysis explanation
-5).
Accompanying drawing 17A and 17B represent the muscle in the animal that CGRP-1 feeds to high lipid and the influence of content of triglyceride.
Accompanying drawing 18 expression CGRP receptor antagonists act on the influence of muscle lipid that high lipid is fed rat to CGRP-1.Shown in result's significance,statistical detect with single factor ANOVA, post-hoc analyzes with Tukey check carrying out, * * * p＜0.001, * p＜0.05 and baseline are relatively;
###Compare with 100nM CGRP p＜0.001;
Compare with 1pM CGRP p＜0.001.
Accompanying drawing 19 expression CGRP-1 to normally feed and the musculus soleus of high lipid feeding animals in the influence of cAMP content.
Accompanying drawing 20 expression perfusion CGRP 1 (100pmol/kg/min) or normal saline 1h, mean arterial pressure descends in the Wistar rat.Mean arterial pressure continues to measure with solid-state pressure transducer, and monitors with the PowerLab/16s data acquisition module.Calibrating signal is presented on the screen, and is kept on the CD with the 2S average of each variable.*P＜0.05。
Accompanying drawing 21 expression perfusion CGRP-1 or the active antagonist of CGRP-1 are to the influence of blood glucose levels.The Advantage Instrument measuring was used in 5 minutes in the every interval of blood glucose levels, and the afterbody blood sample of animal that is poured 90min is as indication.
Invent auspicious stating
I. definition
" lipid decomposition " used herein is meant the enzymatic hydrolysis of lipid or lipid storage chemical compound such as triglyceride, causes free fatty to discharge.Term " free fatty (FFA) " and " deesterify fatty acid (NEFA) " are used interchangeably at this.
" treatment effective dose " used herein is meant the amount of pre-determining of the reagent that calculates the biological or reaction medically be used to eliminate tissue, system, animal or human's class, be that research worker, veterinary, doctor physician or other clinicians seek, as be enough to stimulate lipid to decompose or reduce lipid level and/or improve morbid state or syndromic amount, or prevent, stop, slow down or reverse disease or other syndromic progress of not expecting are to reach the amount of expecting therapeutic effect.
" pharmaceutically acceptable carrier " used herein or " pharmaceutically acceptable excipient " are meant that a kind of disadvantageous body reaction and therapeutic agent of can not causing fully dissolves therein to discharge the carrier of treatment effective dose after using.The example of excipient comprises buffer, normal saline, PBS, dextrose solution, Hank liquid and inert diluent, as calcium carbonate, sodium carbonate, lactic acid, calcium phosphate or dibastic sodium phosphate.
" mammal " used herein has its ordinary meaning, comprises primate (for example human and inhuman primate), experimental animal (for example Rodents such as mice and rat), domestic animal (for example milch cow, boar, sheep and horse) and domestic animal (for example Canis familiaris L. and cat).
Term used herein is meant the prevention of (i) symptom or disease, any clinical symptoms that just eliminates a disease to " treatment " of mammiferous symptom or disease; (ii) suppress symptom or disease, just suppress the development or the progress of clinical symptoms; And/or (iii) mitigation symptoms or disease, clinical symptoms is disappeared.
" EC used herein
50" have a conventional meaning of this area; be meant 50% compound concentration of the maximum facilitation that produces the specific biological effect, for example when the biological agent that be attached to receptor (as high-affinity CGRP receptor) mediation by part (as CGRP) be the concentration of its peaked half.For example, the EC that decomposes by the skeletal muscle lipid of CGRP and high-affinity CGRP receptor for stimulating
50Be to cause 50% the CGRP concentration that promotes with respect to the maximum of baseline values that the skeletal muscle lipid decomposes.The calcium part can be can not be natural also.
Receptor used herein " agonist " is to promote at least a reagent (chemical compound or compositions) that is attached to the relevant biological respinse of receptor usually with the native ligand of receptor.For example, the agonist that is present in the high-affinity CGRP receptor in the Skeletal Muscle Cell is to interact (as combine) with high-affinity receptor and improve the reagent (as CGRP) of the lipid decomposition among the myocyte in dosage dependence mode.Combination between agonist and CGRP receptor can be competed the ability of bind receptor and/or adopt other competition analysiss to determine by agonist and CGRP (as radiolabeled CGRP).
Receptor used herein by " stimulations " or comparably, be " activated " or, comparably by said composition or reagent " excitement ", when said composition or reagent are attached to the metabolism state variation that receptor (as being attached to the ligand-binding site point) causes the cell of expressing this receptor.Normally, for part (as native ligand or other agonist) combination, receptor is given and is transmitted a signal in the cell.When receptor is contacted with specific compound, the generation cell characteristic is the bonded reaction of native ligand (being natural endogenic), and this shows that said composition can stimulate or activate this receptor.Literal coordinate (as activation, stimulation etc.) has equivalent.
" the conservative replacement " used herein, when describing protein, be meant the variation that this proteinic aminoacid is formed, wherein residue is replaced by the structural similarity substitute, and can substantially not change this activity of proteins.Therefore, " the conservative replacement variant " of specific amino acids sequence is meant that those are for the not crucial amino acid whose amino acid surrogates of protein active, the amino acid replacement that perhaps has the aminoacid (as acid, alkalescence, positive charge or negative charge, polarity or nonpolar etc.) of similar characteristic with other is even make the replacement of key amino acid also can substantially not change its activity.It is well known in the art that functional similar amino acid whose conservative replacement table is provided.Following six groups, every group contains the conservative mutually aminoacid that replaces: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) N acid (N) glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); With 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (referring to, Creighton (1984) protein, W.H.Freeman and Company).Those skilled in the art can expect that the above-mentioned replacement of mentioning is not unique possible conservative replacement.For example all charged aminoacid that no matter are positive charge or negative charge can be considered as mutual conservative replacement.In addition, changing on a coded sequence, increase or deleting single amino acids or the amino acid whose indivedual replacements of small scale, disappearance or add also can be " the conservative variant that replaces ".
Term " peptide " is meant the polypeptide of length less than 50 residues.Term " protein " and " polypeptide " comprise " peptide " and long amino acid polymer.
Term " contact " has its ordinary meaning that produces similarity.The method that chemical compound contact with the receptor of being expressed by cell or tissue includes, without being limited to outside body (as external) with cell or tissue and chemical compound is cultivated and make circulation or the other system of chemical compound by animal be and pass cell or tissue to animal compound administration under the condition of this chemical compound energy bind receptor.In some cases, the reagent that is applied is converted into agonist (as the metabolite of the reagent that is applied is a kind of agonist) by the metabolic activity of animal or tissue.
Term used herein " analog of peptide " or " plan peptide " (Fauchere, 1986, Adv.DrugRes.15:29; Veber and Freidinger, 1985, TINS, 392; With Evans etc., 1987, J.Med.Chem30:1229) have its meaning common in this area, refer to as having the analog that is used for the peptide of pharmaceuticals industry with the non-peptide medicine of template peptide similar characteristic.Peptide analogues is usually by calculating molecule modeling exploitation.Structure can be used for producing equal treatment or preventive effect to the analog that the similar peptide of useful peptide is gone up in treatment.Usually, peptide analogues and example polypeptide (polypeptide that promptly has biological or pharmaceutical active) structural similarity, for example natural CGRP polypeptide has one or more peptides connections and preferably is selected from :-CH
2NH-,-CH
2S-,-CH
2-CH
2-,-CH.dbd.CH-(cis and trans) ,-COCH
2-,-CH (OH) CH
2-and-CH
2The connection of SO-replaces, and by method well known in the art and that further describe in following document: Spatola, A.F. is at " aminoacid, peptide and protein chemistry and biochemistry ", B.Weinstein edits, Marcel Dekker, New York, 267 (1983); Spatola, 1983, Vega Data 1:3, " peptide backbone modification "; Morley, 1980, pharmaceutical science trend (TrendsPharm Sci), 463-468; Hudson, D. etc., 1979, Int J Pept Prot Res 4:177-185 (CH
2NH-,-CH
2-CH
2-); Spatola etc., 1986, life sciences (Life Sci) 38:1243-1249 (CH
2-S); Hann, M.M., J Chem Soc Perkin Trans I (1982) 307-314 (CH-CH-, cis and trans); Almquist, R.G. etc., journal of medicinal chemistry (J Med Chem) (1980) 23:1392-1398 (OCH
2-); Jennings-White etc., Tetrahedron Lett (1982) 23:2533 (OCH
2-); Szelke etc., European App.EP45665 (1982) CA:97:39405 (1982) (CH (OH) CH
2-); Holladay, M.W. etc., TetrahedronLett (1983) 24:4401-4404 (C (OH) CH
2-); And Hruby, V.J., life sciences (1982) 31:189-199 (CH
2-S-).The analog of these peptides has than the more significant advantage of polypeptide example, comprise, as: produce more economical, chemical stability is higher, pharmacy characteristic (half-life, absorbability, tire and effect etc.) is better, specificity (as the broad-spectrum biological activity) change, antigenicity reduction etc.The chemical compound of peptide analogues also comprises amino acid whose biology of coordinate, as amino acid whose sulphonyl and boronation analog.
When the lipid content aspect that is used for skeletal muscle, term used herein " normalization " is meant the lipid content in the muscle that reduces the high unusually individuality of muscle triglyceride levels, for example, and the individuality of from the reduction of muscle triglyceride, benefiting.
Term used herein " minimizes ", the time spent of doing when relating to the metabolic response (as blood glucose and lactate level rise) of chemical compound, be meant that chemical compound increases the effect by the metabolic response of amylopectin metabolism receptor for stimulating indistinctively by stimulating high-affinity receptor to mediate the decomposition of enhancing lipid to mammal, tissue or cell.For example, in one embodiment, term " stimulation vasodilation ", " stimulating side effect " or " increasing blood lactic acid " or the like with minimizing with minimizing with minimizing, be meant a kind of or some reagent, its [lipid decomposes to be increased]/[blood glucose increase/blood lactic acid rising/vasodilation enhancing/side effect increase etc.] ratio stimulates the ratio that produces greater than the CGRP-1 (as 200nM) by metabolism amylopectin receptor saturation capacity.Usually this ratio at least about high 2 times, usually at least about high 4 times, sometimes at least about high 10 times).These terms also comprise the reagent or the dosage (being that measured value approaches zero) of the remarkable increase that does not detect vasodilation and/or blood lactic acid and/or other side effect.
II. describe
Foreword
Calcitonin-gene-related peptide (CGRP) is 37 amino acid whose members of the calcitonin family of peptide hormone.CGRP is present in the efferent neuron that contains acetylcholine of the motor end plate of arranging skeletal muscle, and it is released in the synaptic space after exciting nerve.CGRP is found with two kinds of principal modes in the mankind at least, is called CGRP-1 and CGRP-2 (table 1).
Table 1
The basic acid sequence of CGRP-1 of people and rat and CGRP-2 ammonia
hCGRP-1?Ala?Cys?Asp?Thr?Ala?Thr?Cys?Val?Thr?His
Arg?Leu?Ala?Gly?Leu?Leu?Ser?Arg?Ser?Gly
Gly?Val?Val?Lys?Asn?Asn?Phe?Val?Pro?Thr
Asn?Val?Gly?Ser?Lys?Ala?Phe
hCGRP-2?Ala?Cys?Asn?Thr?Ala?Thr?Cys?Val?Thr?His
Arg?Leu?Ala?Gly?Leu?Leu?Ser?Arg?Ser?Gly
Gly?Met?Val?Lys?Ser?Asn?Phe?Val?Pro?Thr
Asn?Val?Gly?Ser?Lys?Ala?Phe
rCGRP-1?Ser?Cys?Asn?Thr?Ala?Thr?Cys?Val?Thr?His
Arg?Leu?Ala?Gly?Leu?Leu?Ser?Arg?Ser?Gly
Gly?Val?Val?Lys?Asp?Asn?Phe?Val?Pro?Thr
Asn?Val?Gly?Ser?Glu?Ala?Phe
rCGRP-1?Ser?Cys?Asn?Thr?Ala?Thr?Cys?Val?Thr?His
Arg?Leu?Ala?Gly?Leu?Leu?Ser?Arg?Ser?Gly
Gly?Val?Val?Lys?Asp?Asn?Phe?Val?Pro?Thr
Asn?Val?Gly?Ser?Lys?Ala?Phe
CGRP and protein amylopectin have about 50% sequence similarity (Cooper etc., 1987, compilation (the Proc Natl Acad Sci USA) 84:8628-32 of NAS; Cooper etc., 1994, endocrine research (Endocr Res.) 15:163-201), but and the biological activity of these two kinds of protein is overlapping differently (sees, as Muff etc., 1985, European endocrinology magazine (Eur.J.Endocrinol.).In its activity, CGRP has significant cardiovascular effect, when comprising the change of vasodilation and forward and inotropic action (Nuki etc., 1993, biochemistry biophysical studies communication (Biochem Biophys Res Commun) 196:245-51; Muff etc., the same; Cooper etc., 1994, endocrine research (Endocr Res.) 15:163-201; Gardiner, 1991, diabetes (Diabetes) 40:948-951; And Takenaga, 1999, European pharmaceutical journal (Euro.J.Pharma.) 367:239-245).In addition, when being applied with high concentration, CCRP is in the news and can causes insulin resistant (see as Leighton and Cooper, 1988, natural 335:632-35).Having proposed insulin resistant should be by suppressing that CGRP discharges or activity be treated and (seen as United States Patent (USP) 6,004,961; United States Patent (USP) 5,641,744).Blood plasma amylopectin and CGRP level raise, and be relevant with insulin resistant in rat, relevant with type 2 diabetes mellitus with obesity in the mankind.In muscle, amylopectin and CGRP have been in the news can be synthetic and promote glycogenolysis to suppress glucose to raise by suppressing glycogen, opposite with the effect as the insulin of non-competing functional antagonist.The peripheral lactose of the high concentration that glycogenolysis produces causes excessive hepatic glycogen to generate as the gluconeogenetic substrate of liver.
The present invention partly relates to CGRP-1 stimulates lipid decomposition (promptly causing triacylglycerol (triglyceride) to reduce) and Mus CGRP-2 in skeletal muscle and the liver to be proved the discovery that the lipid in the stimulation skeletal muscle decomposes.Can detect this reduction from the increase of fatty acid.Shown in embodiment hereinafter, the dose-response that skeletal muscle is exposed to various content rat CGRP-1 the analysis showed that two stages of muscle free fatty acid levels increase, phase I is observed when skin rubs the CGRP-1 of concentration, and second stage is at nanomole concentration C GRP-1 and observed in 2 o'clock.Need not in conjunction with specific mechanisms, these data show that influence that CGRP-1 decomposes the potential lipid of muscle lipid is by the high-affinity CGRP receptor (EC that works
50About 2.6 * 10
-13M[is as 10
-11M to 10
-13In the scope of M]).Therefore, the cell characteristic of expression high-affinity receptor is the CGRP-1 corresponding to nanomole concentration.This concentration is significantly less than to be eliminated insulin resistant and suppresses the synthetic concentration of glycogen (Leighton etc., 1988, natural 335:632-635 in stripped muscle prepared product; Leighton etc., 1989, FEBS Lett 249:357-361).The second stage that lipid decomposes occurs in CGRP-1 and 2 o'clock (EC of higher concentration
50About 4.5 * 10
-8M[is as 10
-9M to 10
-8In the scope of M]), and receptor-mediated by the metabolism amylopectin, promptly can be by the single receptor of CGRP and amylopectin transduction signal in skeletal muscle, it can be used as the receptor (being CGRP/ amylopectin receptor) of these two kinds of peptides.
As describing in further detail hereinafter, the dosage that is characterized as of high-affinity CGRP receptor stimulating agent relies on ground and stimulates the lipid in the skeletal muscle to decompose, and wherein costimulatory receptor mediation and CGRP-1 are with nanomole EC
50With this receptor effect.Metabolism amylopectin receptor stimulating agent is characterized as by the lipid in the receptor-mediated stimulation skeletal muscle and decomposes, and CGRP-1 or 2 is with nanomole EC
50With receptor acting, and amylopectin causes that by receptor lipid decomposes.
Having the metabolism amylopectin passed through calcitonin receptor (oppositely infix form) [C1 by the receptor of body characteristics
Ins-] make up to L6 sarcoplast or cos-7 cell with the ramp-1 cotransfection and (to see embodiment, hereinafter).This receptor has set up that intensive specificity with CGRP, amylopectin and sCT combines and CGRP and the conduction of amylopectin earth signal.The high-affinity binding site of CGRP-1 in muscle and the liver cell is described.Galeazza etc., 1991, peptide 12:585-91 describes two CGRP-1 binding sites in the rat skeletal muscle film preparation thing, has the derivation Kd value of about 37pM and 6nM.Galeazza etc. have also described two CGRP specific binding sites on the rat liver film, have the derivation Kd value of about 44pM and 27nM.Combining of the film fragment of radiolabeled CGRP-1 and liver nonparenchymal cell (as endotheliocyte, lipid torage cell and smooth muscle cell), and not combining with hepatocyte, by Stephens etc., 1991, diabetes, 40:395-400 report.Also can be referring to Poyner etc., 2002, pharmaceutical research 54:233-46.
Lipid decomposes can pass through the receptor-mediated discovery of high-affinity CGRP, and other discoveries at this detailed description provide new being used for to stimulate lipid to decompose and the method and the reagent of the accumulation of lipid of reduction cell and tissue.Especially, the invention provides the lipid that is used for showing skeletal muscle and liver reduces and is used for the treatment of method and the reagent that is characterized as the disease of accumulation of lipid in cell and the tissue.Accumulation of lipid in the skeletal muscle is the Developmental and Metabolic Disorder that shows the key that produces insulin resistant.For example, as if in the relevant insulin resistant of obesity, the metabolic capacity of skeletal muscle is adjusted to lipid esterification rather than oxidation, and the weight loss that diet causes can not correct this deposition (Simoneau etc., 1999, FASEB J.13:2051-60).Also show on evidence muscle lipid normalization be accompanied by insulin sensitivity improve (see as, McGarry etc., 1992, science 258:766-70; Ellis etc., 2000, U.S. physiology endocrine metabolism (AmJ Physiol Endocrinol Metab) 279:E554-60; Oakes etc., 1997, diabetes 46:1768-74; Furler etc., 1997, metabolism 46:1101-6; Kraegen etc., 1991, diabetes 40:1397-403; Dobbins etc., 2001, diabetes 50:123-30; Unger etc., 2000, international fat relevant Developmental and Metabolic Disorder magazine (Int J Obs Relat Metab Disord) 24 appendix 4:S28-32; Manco etc., 2000, metabolism 49:220-4; Kelley etc., 1999, physiology 277 (6Pt1): E1130-41).
The disease and the symptom that are characterized as accumulation of lipid in the skeletal muscle comprise insulin resistant and related symptoms, as the symptom (as type 2 diabetes mellitus, hypertension, obesity, dyslipidemia, arteriosclerosis and thrombosis) and the polycystic ovary syndrome (PCOS) of X syndrome.The disease and the symptom that are characterized as accumulation of lipid in the liver comprise lipid liver (degeneration of liver lipid).The degeneration of liver lipid produces in the patient of solvent damage (as being caused by carbon tetrachloride) or long-term excessive edible ethanol, obese patient and as the result who takes medicine such as glucocorticoid and synthetic estrogen.On the one hand, the invention provides method and the reagent that is used for the treatment of these or other symptom.
The present invention also provides definite and develops the method that is used at the pharmaceutical composition of muscle or the decomposition of its hetero-organization moderate stimulation lipid.
In addition, observed and in mice, knock out the CGRP-1 gene and do not influence vasodilative regulation and control (Lu etc., 1999, molecular cell neuroscience (Mol.Cell.Neuroscience) 14:99-120).Therefore, on the one hand, the invention provides and use, and minimize or avoid to follow usually the side effect of taking heavy dose of CGRP (as vasodilation usually by the method for the agonist treatment insulin resistant of the bonded high-affinity CGRP of CGRP-1 receptor; Acute cardiovascular).Can also realize the beneficial effect that lipid decomposes, minimize the increase (as producing) and the blood-glucose rising (generating) of peripheral lactic acid simultaneously as the liver glucose that produces free lactic acid stimulation from the glycogen degraded.
B. organize lipid content to reduce and to having benefited from the treatment that lipid level reduces the patient of (as in skeletal muscle and/or liver)
On the one hand, the invention provides by organizing or cell (as skeletal muscle or liver) contacts the method that reduces the lipid content in this tissue or the cell with high-affinity CGRP receptor or metabolism amylopectin receptor stimulating agent.Aspect preferred, the invention provides by tissue or cell are contacted the method that reduces the lipid content in skeletal muscle or liver or Skeletal Muscle Cell or the liver cell with high-affinity CGRP receptor stimulating agent.As noted above, this reduction is accompanied by various beneficial effects.In one embodiment, be applied to animal and be generally mammal, tissue or cell are contacted with this agonist by falling agonist.
The invention provides by using the mammiferous method that high-affinity CGRP receptor stimulating agent is treated needs to reduce lipid content (as skeletal muscle or liver), improve the syndrome of the symptom relevant with the muscle accumulation of lipid or reduce the probability that takes place, this symptom is particularly including insulin resistant and " X syndrome " complex (as 2-type diabetes, hypertension, obesity, dyslipidemia, arteriosclerosis and thrombosis), polycystic ovary syndrome and the degeneration of liver lipid.In one embodiment, agonist is the human CGRP, as human CGRP-1.In one embodiment, the agonist of high-affinity CGRP can not substantially stimulate metabolism amylopectin receptor.In the embodiment of this or additive method of the present invention, mammal can be human, primates or non-human primate, or the non-human animal.
In related fields, the invention provides treatment need reduce the syndrome of improving the symptom relevant with the muscle accumulation of lipid of lipid content (as skeletal muscle or liver) or reduce the mammal of the probability that takes place such as people's method, determine to have the symptom relevant by (1) animal of (as insulin resistant, 2-type diabetes, hypertension, obesity, dyslipidemia, arteriosclerosis, thrombosis, polycystic ovary syndrome or the degeneration of liver lipid) with accumulation of lipid, (2) lipid of using in high-affinity CGRP receptor stimulating agent and (3) monitor animal decomposes.In one embodiment, this symptom is an insulin resistant.In one embodiment, this symptom is a type 2 diabetes mellitus.In one embodiment, this mammal is an insulin resistant, but does not have 2-type diabetes.In one embodiment, this mammal insulinize not.Adopt diagnostic method well known in the art (being included in the method for hereinafter describing) to realize easily determining, also can realize with definite method (as gene expression atlas) of knowing any future or developing to suffering from the mammiferous of above-mentioned symptom.Typical high-affinity CGRP receptor stimulating agent behaviour CGRP-1 and its functional variant biologically.In one embodiment, use agonist and can substantially not stimulate metabolism amylopectin receptor.
In one aspect, the invention provides high-affinity CGRP receptor stimulating agent and be used for reducing purposes in patient's the medicament of lipid level in processing or preparation, this patient has benefited from this reduction.On the one hand, the invention provides high-affinity CGRP receptor stimulating agent in the purposes of processing or preparing in the medicament that is used for the treatment of the patient who suffers from X syndrome syndrome, insulin resistant, type 2 diabetes mellitus, hypertension, obesity, dyslipidemia, arteriosclerosis and thrombosis.
In related fields, the invention provides the pharmaceutical composition in unit dose form that contains high-affinity receptor agonist described herein.For example, in one embodiment, when being applied to the mammal such as the mankind, this unit dose produces the agonist of blood or blood plasma level, and this level is lower than the EC of the activator of metabolism amylopectin receptor
50Usually, the agonist of this reagent of blood or blood plasma level is with about 10
-15M is to about 1
-10M exists.For CGRP-1, its blood plasma level may be lower than 300pM and (see embodiment, hereinafter)
The receptor stimulating agent (as the CGRP receptor stimulating agent of high-affinity) that is fit to can be any in all ingredients of hereinafter describing in detail, comprises natural CGRP polypeptide (as human CGRP-1 polypeptide), CGRP polypeptide variants (as functional variant biologically) or non-peptide reagent.
The CGRP receptor stimulating agent is administered to patient's (be body in contact) or body outside, tissue or cell is contacted with agonist, can cause to detect the reduction of lipid content and follow the free fatty increase.It is generally acknowledged that it is the result that lipid decomposes in irritation cell such as the Skeletal Muscle Cell that lipid content reduces.See as embodiment 17 hereinafter.But the applicant is not intended to be subjected to the constraint of any specific mechanisms.Therefore, this paper adopt sometimes statement " lipid decompose stimulation " be convenient for the purpose of, it comprises that lipid level descends and the free fatty increase in the tissue, and does not consider the mechanism that rises and descend.The CGRP receptor stimulating agent stimulates lipid to decompose can be from tissue (as skeletal muscle or liver or blood or blood plasma) to detect in the variation of the content of free fatty acid and composition.Usually, the level that stimulates lipid to decompose is 2 times that the free fatty increase of tissue or blood is at least baseline (level when lacking agonist), at least 4 times and at least 6 times or higher sometimes usually.Detecting the method for free fatty acid levels variation knows.For example, as describing among the embodiment 2 hereinafter, can carry out the gas chromatographic analysis of muscular tissue (as deriving from vivisection or tissue culture).Additive method comprises the HPLC operation of the standard separator of the special fatty acid that is used to prepare structure and metabolic analysis.This method can be used for volatile material, as short-chain fatty acid, is used to study isotope-labeled fatty acid, or is used for separation point position and conformer.The fatty acid of deriving is monitored with the UV spectrophotometer or by exometer (Rao etc., 1995, chromatographic science magazine (J Chromatogr Sci) 33:9-21) usually.Can be by the plasma concentration (Wako Pure Chemical Industries, Osaka, Japan) of determining non-esterified free fatty based on the colorimetric estimation method of S-acetyl-coenzyme-A.Can measure plasma triglyceride (Triglyceride Produre 336, Sigma Diagnostics) with colorimetric determination.Can measure by solvent extraction long-chain coenzyme A, PHASE SEPARATION from tissue with by reverse high performance liquid chromatography and organize the long-chain coenzyme A.
On the one hand, the invention provides high-affinity CGRP receptor stimulating agent is used for reducing patient's lipid level in processing or preparation the purposes of medicament, this patient will have benefited from this reduction.On the one hand, the invention provides high-affinity CGRP receptor stimulating agent be used for the treatment of in processing or preparation suffer from X syndrome, the purposes in the patient's of insulin resistant, type 2 diabetes mellitus, hypertension, obesity, dyslipidemia, arteriosclerosis and thrombosis the medicament.
Has special meaning although activate the reagent of (agonize) high-affinity receptor, on the other hand, the invention provides the mammiferous method that treatment need reduce lipid content, comprise and use metabolism amylopectin receptor but not the agonist of high-affinity receptor stimulates the lipid in the mammal to decompose, realize this result by using this metabolism receptor stimulating agent with the pharmaceutical composition that contains this agonist, descending owing to lipid decomposes with treatment needs the mammiferous method of this treatment.In one embodiment, agonist is rat CGRP-2.In one embodiment, agonist is the receptor for stimulating variant of CGRP.In one embodiment, agonist is not an amylopectin.In another embodiment, agonist is an amylopectin.The application process of this agonist and mode change on dosage to described herein to be used for the method that the high-affinity receptor agonist uses similar with mode, and this is obvious for the technical staff.
C. high-affinity CGRP receptor stimulating agent
Used high-affinity CGRP receptor stimulating agent comprises various types of compounds in the present invention's practice, comprises the analog and the non-peptide compound of peptide, peptide.Suitable agonist is described hereinafter and/or can be definite with method described herein (and/or by because the present invention openly instructs those of ordinary skill be obvious method).High-affinity CGRP receptor stimulating agent stimulates the lipid in the skeletal muscle to decompose.Preferred agonist and/or dosage cause preferably stimulating high-affinity CGRP receptor with respect to metabolism amylopectin receptor.
The preferred stimulation
Point out that as this paper some tissue (as skeletal muscle) is expressed high-affinity receptor and metabolism amylopectin receptor, and some chemical compound such as rat CGRP-1 are the agonist of high-affinity receptor and metabolism amylopectin receptor.As noted above, the lipid decomposition can be receptor-mediated by these two kinds.But open as this paper, expectation preferably stimulates high-affinity receptor usually." the preferred stimulation " has this term conventional meaning, can be described (or detection) in every way.The EC that has this high-affinity receptor when the high-affinity receptor agonist
50Shi Fasheng " the preferred stimulation ", this EC
50Be lower than the EC of metabolism amylopectin
50, the dosage of this agonist and concentration are lower than the EC of metabolism amylopectin receptor
50, but be equal to or higher than the EC of high-affinity receptor
50In one embodiment, when the high-affinity receptor agonist does not cause any detectable stimulation to metabolism amylopectin receptor, realize " the preferred stimulation " (for example at any dosage, any be lower than 10 μ M or any 10mM of being lower than).
Therefore, in one embodiment of the invention, high-affinity CGRP receptor stimulating agent preferably stimulates the amount of high-affinity CGRP receptor to be applied to produce with respect to metabolism amylopectin receptor.This preferred stimulation can be implemented, for example by adjusting the dosage or the prescription of agonist, perhaps according to the characteristic of agonist.
In a preferred embodiment, relative metabolism amylopectin receptor contacts the reagent of the preferred stimulation of the high-affinity CGRP receptor preferred stimulation by the cell participant being caused this high-affinity CGRP receptor and to realize.In one embodiment, this reagent is the agonist of high-affinity CGRP receptor, and can substantially not stimulate metabolism amylopectin receptor.In one embodiment, this reagent has an EC who influences lipid decomposition stimulation by metabolism amylopectin receptor
50, it is significantly greater than influencing the EC that lipid separately wins stimulation by high-affinity CGRP receptor
50In one embodiment, skeletal muscle is contacted the generation (measuring as the state (appearance) that passes through FFA in tissue or the blood plasma) that causes more FFA with this reagent, this is because the lipid of high-affinity receptor decomposes the lipid decomposition stimulation of stimulation greater than metabolism amylopectin receptor.Can expect that whether the effect of determining a kind of reagent is that to stimulate the result's of high-affinity CGRP method be that known amylopectin is to work by the metabolism receptor by the relatively effect of this reagent and the effect (having the dose-effect curve similar to CGRP) of amylopectin.
In some embodiments, this reagent influences the EC that lipid decomposes to stimulate by metabolism amylopectin receptor
50Significantly influence the EC that lipid decomposes to stimulate by high-affinity CGRP receptor greater than this reagent
50, it is for the EC of high-affinity receptor
50Than the EC that is used for metabolism amylopectin receptor
50At least low 10 times, more generally low at least about 100 times, at least about low 500 times, at least about low 1000 times, at least about low 5000 times, at least about low 10000 times.But, more generally, the EC of high-affinity CGRP receptor
50At least about low 10
5Doubly, at least about low 10
6Doubly, at least about low 10
7Doubly, at least about low 10
8Doubly.Be described as the EC of low affinity receptor
50(" EC
50-amylopectin-R") with the EC of high-affinity receptor
50(" EC
50-CGRP-R") ratio, i.e. EC
50-amylopectin-R/ EC5
0-CGRP-R, for having the higher significantly EC that stimulates by high-affinity receptor
50Reagent, this ratio is usually at least about 10, usually at least about greater than 10
2, greater than 5 * 10
2, greater than 10
3, greater than 5 * 10
3, greater than 10
4, greater than 10
5, greater than 10
6, greater than 10
7, or greater than 10
8Be used to measure the EC of chemical compound
50Method be known, describe hereinafter.
In some embodiments, when contacting when being applied or with cell or tissue, high-affinity CGRP receptor stimulating agent does not have any remarkable or any detectable stimulating activity to metabolism amylopectin receptor.The active detection of significant stimulation to metabolism amylopectin receptor is when pouring into agonist to animal (as rat), produces with respect to perfusion normal saline or the control animal blood lactic acid of other non-active ingredients or the remarkable increase of blood-glucose.Significance is to determine by intrinsic average and standard variance in conventional method such as the measured value.Significant difference available standards analytical method between contrast and the processing method such as t-check the check of significance ground.Usually, adopt n＞1 to analyze, as be at least 7 and be used for bigger statistical edge.To lactic acid generate to increase and skeletal muscle in the analysis that raises of glucose by metabolism amylopectin receptor antagonist insulin stimulating be well known in the art (see as Cooper, 1994, endocrinology review (Endocr Rev) 15:163-201; Young etc., 1993, FEBSLett.334 (3) 317-321; With Young etc., amylopectin is active to detect US6,048,514).
Can choose cell and tissue, carry out other detections from cell and tissue, cell line and the reconstitution cell of other mammals (as mice and rat).Detection comprises cell detection, extra-organismal detection (as the isolated rat musculus soleus) and integral animal is studied.For example, can carry out cell analysis with the recombinant receptor of describing among the embodiment hereinafter based on cell.
In a kind of analysis, use the isolated rat musculus soleus.Rat soleus muscle contains high-affinity CGRP receptor and metabolism amylopectin receptor, and the isolated rat musculus soleus is specially adapted to distinguish to the not same-action of isoacceptor not.Musculus soleus can derive from the animal of high lipid or conventional food of feeding, make the dose-response curve of chemical compound, the reflection of the process that wherein generates analysis reflection (reflective) (as mensuration of muscle free fatty and content of triglyceride) of the process that (i) take place by high-affinity CGRP receptor and metabolism amylopectin receptor and (ii) take place by metabolism amylopectin receptor (as the muscle glycogen content when having strong stimulation insulin [
14C]-conversion of glucose is the mensuration of glycogen).Shown that amylopectin has similar effect with CGRP to glycogen metabolism, especially, to the conversion of glucose of insulin stimulating be glycogen noncompetitive inhibition (analyze as an example, see as Muff etc., the same; Copper etc., 1994, endocrinology is looked back 15:163-201; Gardiner, 1991, diabetes 40:948-951; Takenaga, 1999, European pharmaceutical journal (Euro.J, Pharma.) 367:239-245; Leighton etc., 1988, natural 335:632-5; Cooper etc., 1988, the compilation 85:7763-6 of NAS).A kind of reagent, dosage or prescription produce expression to have stimulated high-affinity CGRP receptor and not to have produced result to the indication of metabolism amylopectin receptor-specific sexual stimulus, then is considered to not the agonist of high-affinity CGRP receptor that can substantial activation metabolism amylopectin receptor.In one embodiment, distinguish by the selective antagonist of high-affinity and/or metabolic receptor with effect metabolic receptor high-affinity.With respect to control tissue, cell or animal (as not being exposed in the agonist), do not cause the reagent of metabolism amylopectin receptor active significant change or the activity that processing (decomposing enhancing as receptor-mediated lipid) is considered to substantially not stimulate metabolism amylopectin receptor.This paper also provides other detections (as seeing embodiment) of the preferred stimulation of determining high-affinity receptor.
As noted above, in one embodiment of the invention, the agonist of high-affinity receptor does not substantially stimulate the dosage of low affinity muscle metabolism amylopectin receptor to be applied to mammal (or contact with in vitro tissue) with the high-affinity CGRP receptor of the skeletal muscle that is enough to stimulation of host.Therefore, a kind of reagent stimulates high-affinity receptor and metabolism amylopectin receptor when high concentration, the dosage that can use by adjustment (as, adjust prescription make only have the reactive compound of tested measurement to be released and can and receptor acting) regulate any stimulation maximization of the effect of this reagent with respect to metabolism amylopectin receptor." dosage that is applied " used herein is intended to comprise that tissue and reagent are in the body outer contacting.
The preferred dose scope that the high-affinity receptor agonist is applied to mammal (as the mankind) descends at least about 15% minimum dose for cause lipid in skeletal muscle or liver, preferably at least about 25%, more preferably at least about 30%, more preferably at least about 50%, or higher, 75% (as measuring) according to appointment by the variation of free fatty or triglyceride levels.
Another preferred dose scope that the high-affinity receptor agonist is applied to mammal (as the mankind) is to produce the content of the stimulation that lipid is decomposed at mammalian bone flesh, and does not stimulate or Min. ground stimulation vasodilation.This content can be measured by many methods, carries out the dose-response analysis for experimental animal (as by I.V., oral or other approach of I.P.) as the CGRP receptor agonism agent prescription of using a large amount of various dose by (i); (ii) detect the influence that each dosage decomposes the lipid in the experimental animal muscle and detect each dosage, decompose and vasodilative dose response data thereby set up lipid to the vasodilative influence of experimental animal; (iii) determine the dosage of CGRP receptor agonism agent prescription from the dose response data, it improves the lipid decomposition and increases vasodilation indistinctively in mammal, or only Min. ground increases vasodilation.Vasodilation can be with any mensuration in the various detection methods (seeing as Nuki etc., 1993, biochemistry biophysical studies communication (Biochem Biophys Res Commun) 196:245-51).In the mankind, vasodilation can be monitored by measuring systolic pressure and mean arterial blood pressure.
Another preferred dose scope that the high-affinity receptor agonist is applied to mammal (as the mankind) is to produce the stimulation that lipid is decomposed at mammalian bone flesh, and does not stimulate or Min. ground stimulates the raise content of (being to raise significantly on the statistics of relative baseline values) of glucose in the blood and/or lactate level.This content can be measured by many methods, carries out the dose-response analysis for experimental animal (as by I.V., oral or other approach of I.P.) as the CGRP receptor agonism agent prescription of using a large amount of various dose by (i); (ii) detect the influence that each dosage decomposes the lipid in the experimental animal muscle and detect the influence of each dosage to experimental animal glucose and/or lactate level, lipid decomposes and the dose response data of glucose/lactate level thereby set up; (iii) determine the dosage of CGRP receptor agonism agent prescription from the dose response data, it strengthens the lipid decomposition and does not improve or Min. ground raising glucose and/or lactate level in mammal.Blood-glucose and lactic acid can be measured with conventional method, as by being present in immobilized enzyme chemistry (glucoseoxidase, L-Lactate Oxidase, analyser model 2300-STAT, the Yellow SpringsInstruments in the standard glucose analyser, Yellow Springs, the Ohio).In brief, oxidized when substrate enters the enzyme layer, generate hydrogen peroxide, hydrogen peroxide sees through cellulose acetate to platinum electrode and oxidized.The electric current and the concentration of substrate that produce are proportional.Also can be referring to as Ye etc., 2001, diabetes (Diabetes), 50:411-417; Ellis etc., 2000, U.S. physiology magazine (Am.J.Phys.) 279:E544-E560.
When being natural CGRP-1 polypeptide (as human CGRP-1), be created in the EC of high-affinity CGRP receptor
50In the scope (as from 10
-11M to 10
-13The M scope) is lower than the EC of metabolism amylopectin receptor
50(as from 10
-9M to 10
-8The M scope) pharmaceutical formulation of blood or serum-concentration CGRP-1 is useful especially.For CGRP-1 and functional variant thereof, typical doses generation blood plasma or serum levels are about 10
-15M is to about 10
-10Between the M, about 10
-15M is to about 10
-11Between the M, or about 10
-15M is to about 10
-12Between the M.Useful blood plasma or serum levels are usually＜10
-10M.These scopes are appreciated that for example rather than are used for limiting.The blood plasma and the serum levels of chemical compound (comprising non-peptide compound) can be measured by conventional method, as ELISA, RIA, spectrophotography, enzymatic analysis or additive method).
Be appreciated that by the instruction of this paper and guide that the whole bag of tricks can be used to determine preferably to activate or stimulate with respect to metabolism amylopectin receptor chemical compound, compositions, dosage and the prescription of high-affinity CGRP receptor.Obviously these methods can be used to screen the reagent that is used for the treatment of insulin resistant and other symptoms.
Adopt the competition of recombinant receptor to combine affinity (as K with the inherence of metabolism amylopectin receptor in conjunction with detecting to produce about chemical compound
iValue) information, and confirm whether target compound works by receptor-mediated mechanism.For example, if target compound can generate the blocking-up second message,second messenger of metabolism amylopectin receptor place by amylopectin or CGRP, just this shows that this chemical compound is a kind of antagonist so.If when lacking amylopectin or CGRP, chemical compound itself can cause that the second message,second messenger reacts, this shows that the chemical compound that is studied is a kind of agonist so.About the information of chemical compound relative potency, as pass through EC
50Parametric measurement obtains from second message,second messenger (as cAMP) reaction under being enough to the compound concentration of saturated this receptor.Can determine suitable concentration in conjunction with affinity according to the inherence of chemical compound.
The example agonist
All cpds can activate high-affinity CGRP receptor, comprises polypeptide and intends peptide and non-peptide compound such as little organic molecule (as molecular weight＜1000).
In one embodiment, high-affinity CGRP receptor stimulating agent is a polypeptide.In one embodiment, this polypeptide has and identical sequence or the extremely similar sequence of natural CGRP-1 polypeptide (as human CGRP-1 polypeptide or derive from the homologue of rat, pig, milch cow, rabbit, chicken, salmon or other species); See Cooper etc., 1994, endocrinology is looked back 15:163-201).In this article, a kind of specific polypeptide has and the extremely similar sequence of natural CGRP-1 polypeptide, when arranging by preferred coupling, this specific polypeptide contain corresponding to natural CGRP-1 polypeptide (as the mankind) at least about 75%, sometimes at least about 80% with sometimes at least about the aminoacid sequence of 90% residue.In one embodiment, the sequence of receptor stimulating agent only is different from CGRP-1 (as human CGRP-1) by conservative the replacement.
In related embodiment, high-affinity CGRP receptor stimulating agent is the functional variant biologically of CGRP-1 polypeptide, as the analog (as peptide analogues) of polypeptide or peptide.Polypeptide is the functional variant biologically (comprising polypeptide and the conservative variant that replaces with significant sequence homogeneity) of CGRP-1 polypeptide, characterized by following characteristic: (i) decompose at skeletal muscle moderate stimulation lipid, (ii) it influences the EC of the stimulation of lipid decomposition by metabolism amylopectin receptor
50Significantly influence the EC of the stimulation of lipid decomposition by high-affinity CGRP receptor greater than it
50Be that it preferably stimulates high-affinity CGRP receptor), usually (iii) when arranging by preferred coupling, contain the sequence length that is equivalent to natural CGRP-1 (as human CGRP-1) variant at least about 60%, sometimes at least about 75%, sometimes at least about 80% with sometimes at least about the aminoacid sequence (promptly extremely similar) of 90% residue.
By instruction disclosed by the invention, can design and detect the activation activity of variant.Traditional mutation method (as method rite-directed mutagenesis, alanine scanning and the analysis that this paper describes or this area is known) can be used to determine the functional variant biologically of CGRP-1.In one aspect, the invention provides the method for non-natural agonist of producing the high-affinity CGRP receptor can be used for preparing therapeutic agent, obtain natural CGRP polypeptide (as human CGRP-1) sequence by (i); (ii) generate the CGRP variant by replacing or deleting at least one amino acid residue of modification; (iii) detecting variant stimulates by receptor-mediated one or more active abilities of high-affinity CGRP (decomposing stimulating activity as the lipid in the skeletal muscle); The variant of (iv) determining preferably to activate high-affinity CGRP receptor with respect to metabolism amylopectin receptor is as non-natural agonist of the high-affinity CGRP receptor that is used to prepare therapeutic combination.
In design and screening high-affinity CGRP receptor stimulating agent, those technology by disclosure instruction it is also understood that, at least the aminoterminal circulus part of natural CGRP is necessary (the flush end CGRP peptide that promptly lacks circulus is the active antagonist of some CGRP) for activity, comprises the high-affinity receptor agonist activity.
2Cys-
7Cys disulfide bond (formation contains 6 amino acid whose " ring-type " structures) and C-terminal amine be in the news be complete biologic activity necessary (Cooper etc., 1988, NAS compilation, 85:7763-66).Therefore, in one embodiment, comprise this seed amino acid circulus as the functional variant biologically of the CGRP-1 of high-affinity CGRP-1 receptor stimulating agent, or coordinate (as the homologue of non-peptide structure), and/or amidatioon.In one embodiment, this agonist is the polypeptide with general formula of the CGRP family concensus sequence that is in the news: Xxx Cys Xxx Thr Ala Thr Cys Val Thr His Arg Leu Ala Xxx XxxLeu Xxx Arg Ser Gly Gly Xxx Xxx Xxx Xxx Asn Phe Val Pro Thr Xxx Val GlyXxx Xxx Ala Phe, wherein Xxx is any aminoacid, see Cooper etc., endocrinology looks back 1994,15:163-201.
In some embodiments, this agonist is or corresponding to the fragment (as at least 6 residues, more frequent is 20,25,30 or 35 residues) of CGRP polypeptide.In one embodiment of the invention, this reagent has the sequence of chimeric CGRP polypeptide, wherein one or more aminoacid are replaced by different aminoacids in a kind of native sequences of species, obtain the corresponding sequence of the CGRP-1 of one or more different species (as rat).
CGRP peptide (as comprise biologically functional variant) can be with these proteinic known arrays by conventional synthetic and recombinant methods.The recombinant technique and the additive method that are used for the present invention practice be known in the art and be described, as Sambrook and Russel (2001) molecular cloning: laboratory manual (third edition) cold spring lane laboratory publishing house; Current method in Ausubel etc. (1987) molecular biology (additional in calendar year 2001) John Wiley﹠amp; Sons, New York.From the beginning chemically synthesized polypeptide be know and can be used for preparing the CGRP polypeptide and (see as Caruthers etc., 1980, Nucleic Acid Res.Symp.Ser., 215-223; With Horn etc., 1980, Nucleic Acid Res.Symp.Ser., 225-232).For example, the synthetic available various solid phase techniques of peptide carry out (Roberge etc., 1995, science 269:202), comprise synthetic automatically (as with Perkin ElmerABI 431A peptide synthesizer, recommending to carry out according to the manufacturer).New synthetic peptide can be by purification fully, as the high performance liquid chroma-tography (as Creighton, protein, structure and molecule mechanism WH Freeman andCo, New York [1983]) by being used to prepare.CGRP amino acid sequence of polypeptide or its any part can be changed in directly synthetic and/or be made up and produce the polypeptide of variation of the present invention with chemical method and other proteinic sequences (more particularly with the homology CGRP of other animal species sequence).In addition, the CGRP polypeptide can separate from natural material.
The nucleic acid of the polynucleotide of CGRP (genome and cDNA) and aminoacid sequence and polypeptide are known, adopt search word " CGRP " and " calcitonin-gene-related peptide (calcitonin gene-related peptide) " can find (as http://www.ncbi.nlm.nih.gov/databse/index.html) in scientific and technical literature and GenBank.The GenBank accession number of CGRP polypeptide is provided in table 2.
Table 2
Proteic GenBank accession number of CGRP and gene
| Species | The GI login |
| Rattus (Rattus rattus) | ???115，281020；203227 |
| House mouse (Mus musculus) | ???12744898，13241720 |
| Jungle fowl belongs to (Gallus gallus) | ???115539；2134304；222802；1334708 |
| Sheep (Sheep) | ???399233；109012；256539 |
| Double-colored blade Jellyfish (Phyllomedusa bicolor) | ???7387567 |
| Class people (Homo sapiens) | ???115482；2144644；179828；296638；9945304；1476 ???2355；155487；86999；9929240；14250056；180466；147 ???68186；2134847；1340176；179799；457134；223948 |
| Common cattle (Bos taurus) | ???227702 |
| European rabbits (Oryctolagus cuniculus) | ???10281574；227771；10281572； |
| The lake frog (Rana ridibunda) | ???399232；388464 |
| Canis (Canis familiaris) | ???9367718 |
| Equus man horse (Equus caballus) | ???7804969；7804971 |
| Pig (Pig) | ???399231 |
| Fish (Oncorhynchus species) | ???231851；1730319 |
Can be expected in the embodiment of method of the present invention and various compositionss, agonist and tested chemical compound and reagent (as being used for screening) are the outer chemical compounds of CGRP.Whether for example the invention provides by detecting a kind of reagent is that high-affinity CGRP receptor stimulating agent determines whether this reagent is used in the method that mammal moderate stimulation lipid decomposes.In an expected embodiment of this method, this reagent is not CGRP.
In one embodiment, this agonist is not the CGRP related polypeptide.For example, this agonist be irrelevant polypeptide of sequence or, in addition, non-peptide molecule.The non-polypeptide agonist of example can be chemical compound such as carbohydrate such as oligosaccharide and polysaccharide; Polynucleotide; Lipid and phospholipid; Fatty acid; Steroid; Dipeptides, amino analog and organic molecule are as micromolecule.In certain embodiments, agonist is not human CGRP-1 or is not mammiferous CGRP-1 polypeptide.
In one embodiment, polypeptide or non-polypeptide agonist prepare with derivation drug design method (as adopting complete methodology, comprising structural analysis, synthetic chemistry and the advanced computational tool of target molecule).See as Kim etc., 2000, unite chemical high flux screening (Comb Chem High Throughput Screen) 3:167-83 and Coldren, 1997, compilation (Proc.Natl.Acad.Sci.USA) 94:6635-40 of NAS).
In one embodiment, agonist is the chemical compound of determining according to screening technique described below.
On the one hand, the invention provides the method for pharmaceutical compositions, by preparing above-mentioned agonist, and this agonist is combined with pharmaceutically acceptable excipient.In some embodiments, this pharmaceutical composition is prepared under the condition with waiting to ooze aseptic, abides by all regulations of the Good Manufacturing Practice (GMP) of FDA (Food and Drug Adminstration) fully.
At different aspect, be attached to the discovery that the metabolism amylopectin is known from experience stimulates lipid to decompose based on CGRP-1, the invention provides by with CGRP-1 or by the variant polypeptide of the CGRP-1 of metabolism amylopectin receptor for stimulating stimulate high-affinity CGRP receptor or metabolism amylopectin receptor or they both, the method that stimulates lipid to decompose.Usually, monitor the step of the variation of the lipid decomposition level in mammalian tissues (as by measuring the amount of the free fatty in mammiferous muscle, liver, blood or its hetero-organization) or the in vitro tissue in addition.Metabolism receptor for stimulating CGRP-1 variant is by following characteristic present: (i) stimulate the lipid in the skeletal muscle to decompose, (ii) when pressing the optimum matching arrangement, they contain corresponding to the length of the sequence of natural CGRP-1 variant such as human CGRP-1 sequence at least about 60%, sometimes at least about 75%, sometimes at least about 80% with sometimes at least about the aminoacid sequence of 90% residue.In one aspect, the invention provides by contacting the method that stimulates the lipid in skeletal muscle or the liver to decompose with mammalian skeleton flesh or liver and CGRP-1 or by its variant of metabolism receptor for stimulating.
On the one hand, the invention provides a kind of therapeutic scheme, comprise that (i) gives and to suffer from or administration CGRP-1 polypeptide, its functional variant or its variant that is stimulated by the metabolism amylopectin and lipid of (ii) monitoring in the mammal biologically that susceptible is characterized as the symptom of accumulation of lipid in the skeletal muscle decompose.
D. agonist uses and dosage form
According to the present invention, in one aspect, the high-affinity CGRP receptor stimulating agent (as the CGRP-1 polypeptide) of treatment effective dose is applied to the patient (as patient or animal), and this patient will have benefited from organizing lipid content (as the skeletal muscle lipid content) to reduce.As hereinafter discussing, this reduction of lipid content is useful to the patient with various symptoms, these symptoms such as insulin resistant, type 2 diabetes mellitus, hypertension, obesity, dyslipidemia, arteriosclerosis and thrombosis.The dosage range of using reagent of the present invention is the scope that is enough to produce desired effects (descends or improve syndrome or symptom development as lipid content).
In related fields, the invention provides the agonist of the high-affinity CGRP receptor of the unit dosage form that is used to be administered to the patient." unit dosage form " used herein is meant and is used for the compositions that single administration is treated the patient who suffers from disease or symptom.Each unit dosage form generally comprises various active component of the present invention and adds pharmaceutically acceptable excipient.The example of unit dosage form is independent tablet, independent capsule, batch powder, aqueous solution, suppository, Emulsion or suspension.The treatment of disease or symptom needs regular administration of unit doses form, and for example, every day is unit dosage form more than 2 times or 2 times, every meal once, per four hours or other are used once at interval, or every day is only once.Statement " oral unit dosage form " expression is designed to the unit dosage form by orally administer.The prescription that is used to inject also can provide with unit dosage form, as in ampoule or multidose container.
Unit dosage form of the present invention contains the receptor stimulating agent for the treatment of effective dose.In one embodiment, the administration of unit doses form produces the certain level agonist in mammal, preferably stimulates high-affinity receptor (with respect to metabolism amylopectin receptor).Therefore, in one embodiment of the invention, the agonist of high-affinity receptor (as CGRP-1 albumen or its functional variant biologically) is applied (as monitoring the known effect that takes place by metabolism amylopectin receptor, as blood or blood plasma lactic acid and glucose level increase) can not make with the saturated concentration that combines of metabolism amylopectin receptor.In one embodiment, this dosage only causes blood or blood plasma lactic acid and glucose level or vasodilative minimal increase (if there is).
Though given dose will depend on the molecular structure and the chemical characteristic of specific agonist, the technical staff of pharmaceutical field can understand the employing routine techniques from this paper open can determine appropriate dosage.For example, the dosage of high-affinity CGRP receptor stimulating agent or prescription can be determined by variety of way, this agonist stimulates the lipid in the mammalian bone flesh to decompose, and not or Min. ground in mammal, cause the side effect do not expected (as, increase mammiferous blood-glucose, blood lactic acid or vasodilation).As using in this article, " level of rising " is meant with respect to the increase that pre-determines level (as the assign thresholds level of side effect).Carry out this definite method and comprise that (i) gives the test mammal by the CGRP receptor stimulating agent that (a) uses a large amount of various dose (or prescription); (b) measure the influence that various dosage or prescription decompose the lipid in the test mammalian tissues and measure influence to side effect, carry out the dose response analysis, lipid decomposes and the dose response data of side effect thereby set up; Determine to increase (ii) that lipid decomposes and the dosage that can not cause the CGRP receptor agonism agent prescription of side effect from the dose response data.In one embodiment, various dosage or prescription are determined by the free fatty acid levels of measuring in the animal tissue the influence that lipid decomposes.Can adopt in vitro tissue to substitute whole animal and carry out similar approach.And based on the instruction of this paper, those of ordinary skill can adopt any the whole bag of tricks to determine the dosage of expectation.
Usually, the dosage of non-peptide compound agonist will be from 10
-16M to 10
-5In the concentration of M (as in one or more muscle, blood, serum or blood plasma, measuring).As noted above, when being CGRP-1 polypeptide (as human CGRP-1), make the blood plasma of CGRP-1 or serum-concentration EC at high-affinity CGRP receptor
50Scope interior (10
-10M to 10
-13M), still be lower than the EC of metabolism amylopectin receptor
50(10
-9M to 10
-8M) dosage is useful especially.For CGRP-1 and functional variant biologically thereof, example dosage is created in about 10
-15M is to about 10
-10Serum between the M or blood plasma level.In one embodiment, serum levels is lower than 300pM.
Reagent be applied to animal produce aspiration level or concentration amount of reagent depend on many factors that the technical staff knows, as chemical compound half-life (as serum half-life) and frequency of using and mode.Be used for explanation rather than restriction, the dosage of CGRP-1 polypeptide is applied with the scope of 20 piks to 1 grams, be more typically every day in 3 milligammas to 50 micrograms.In various embodiments, unit dose (being daily dose sometimes) is lower than about 10 micrograms, less than about 1 microgram, less than about 100 milligammas, less than about 10 milligammas, less than about 1 milligamma, less than about 100 piks with less than about 10 piks.
According to the above-mentioned data that derive from the original doses response curve and other data of obtaining by conventional method, other scopes of polypeptide and non-peptide compound are obvious for experienced practitioner.
The present invention also provides the compositions that contains with the bonded high-affinity CGRP of pharmaceutically acceptable excipient receptor stimulating agent.In one embodiment, this reagent and excipient are formulated into and optionally activate high-affinity CGRP receptor and do not activate metabolism amylopectin receptor.In another embodiment, this reagent and excipient are formulated into optionally stimulates lipid to decompose, and not or Min. ground stimulate disadvantageous side effect, as vasodilation, or blood-glucose or lactate level raise.
(make up individually or with CGRP as reagent, reduce lipid, free fatty and/or long-chain coenzyme A level) can be prepared or be used jointly to CGRP polypeptide or other agonist with other active agents.In one embodiment, agonist of the present invention is not used jointly with insulin.In one aspect, the high-affinity receptor agonist reagent (as antireceptor antibody) that do not suppress high-affinity receptor with suppress activating metabolism amylopectin receptor is used.
The agonist (or antagonist) that uses in the present invention's practice can directly be applied to processed host.Use selectively and under aseptic condition, carry out.But,, preferably be provided with pharmaceutical formulation though active component can be used separately.Prescription typically contains at least a active component and one or more its acceptable carriers.Consider with the compatibility of other compositions and the patient do not damaged that various carriers should be pharmaceutically to go up acceptable with the physiology.Can fill a prescription by the method preparation treatment that any pharmaceutical field is known, see as (editor) (1990) GOODMAN AND GILMAN ' S such as Gilman: therapist's pharmacy basis (ThePharmacological Bases of therapeutics) (the 8th edition) Pergamon publishing house, (1990) Remington, practice and pharmaceutics science (the science of practice and pharmacy), the 20th edition, Mack Publish Co., Eston, P.A.; Avis etc. (editor) (1993) pharmaceutical dosage forms: Dekker parenteral medicine (Parenteral Medications Dekker), New York; Lieberman etc. (editor) (1990) pharmaceutical dosage forms: Dekker tablet (Tablets Dekker), New York; Lieberman etc. (editor) (1990) pharmaceutical dosage forms: Dekker dispersion (Disperse Systems Dekker), New York.
Chemical compound of the present invention can be applied by oral, parenteral (as intramuscular, intraperitoneal, intravenous, ICV, that injection of brain pond or perfusion, subcutaneous injection or heeling-in), the path of using that sucks spraying, nasal cavity, vagina, rectum, Sublingual or body surface, and can be suitable for variously using the path and being formulated in separately or together in the prescription of the optimal dose unit of containing traditional avirulent pharmaceutically acceptable carrier, adjuvant and excipient.When agonist is polypeptide, usually use (as intravenous) by parenteral.
(as keeping specific blood plasma concentration) if desired, agonist can sustained release the prescription form be applied to the patient.Various suitable Controlled Release System are known, comprise being suitable for oral, parenteral and other use the form in path.See as Bogner etc., 1997 U.S.Pharmacist1997; 22 (appendix): 3-12; And manual industry: sustained-release oral preparation form: the exploitation of inside and outside dependency, evaluation and application.Rockville, MD: Food and Drug Admistraton, drug evaluation and research center, 1997.Can in the canonical reference document, find the example of control pharmaceutical formulation (for example, to see: Sweetman, S.C. (editor) Martindale, complete drug reference (Thecomplete Drug Reference) (the 33rd edition), Pharmaceutical Press, Chicago, 2002,2483; Also can be referring to Aulton, M.E. (editor) pharmacopedics, medicine type design science (The Scienceof Dosage FormDesign), Churchill Livingstone, Edinburgh, 2002,734; Can also be referring to Ansel, H.C., Allen, L.V. and Popovich, N.G., pharmaceutical dosage forms and drug delivery system, the 7th edition, Lippincott 1999,676).The excipient that adopts in drug delivery system is introduced in various publications, is (as seeing: Kibbe, E.H. handbook of pharmaceutical excipients, the 3rd edition, american pharmaceutical association, Washington, 2000,655) that those skilled in the art know.USP provides the example of the peroral dosage form of many improvement releases (as seeing American Pharmacopeia 23/ prescription 18 (the United States Pharnacopeia 23/National Formulary 18) of country, The United States PharmacopeialConvention, Inc., Rockville MD, 1995).The tablet and the capsular drug release ability that also provide total chapters and sections and special test to measure slow release and postpone to discharge in the disclosure thing.In one aspect of the invention, this agonist and exercise event are collaborative to be used, and strengthens the patient by tempering the triglyceride reducing effect.
Pharmaceutical composition can be injectable aseptic aqueous solution or oily suspensions.This suspension can be according to known systems, prepares with those suitable dispersants or wetting agent and suspending agent.These injectable sterile preparation things can also be injectable sterile solution or the suspensions that is dissolved in acceptable non-toxicity diluent of parenteral or the solvent.Acceptable excipient that can be used and solvent are water, Ringer ' s solution and isoosmotic sodium chloride solution.But, be to be understood that the given dose level that is used for any particular patient is different with administration frequency, this depends on various factors, comprises the activity of used specific compound, the metabolic stability of this chemical compound and the order of severity of action length, age, body weight, healthy symptom, sex, diet, the mode of using and time, excretion rate, drug regimen and specific symptoms.In certain embodiments, every day or to use agonist weekly be feasible.
E. monitor lipid content, blood glucose levels, blood lactic acid level, vasodilation, insulin resistant and other symptoms
In some embodiments of the invention, the CGRP receptor stimulating agent is applied to mammal, monitors in this mammal or its tissue (mammalian bone flesh) the stimulation of lipid decomposition or the reduction of lipid content.Usually monitoring lipid by the variation that detects dissociate in the destination organization (as skeletal muscle, blood, blood plasma) content of fatty acid or composition decomposes.Detecting the method for free fatty acid levels variation knows.For example, as hereinafter describing among the embodiment 1, can carry out the gas chromatographic analysis of muscular tissue (deriving from vivisection or tissue culture).Additive method comprises by measuring the non-esterified fatty acid of blood plasma (NEFA) based on the oxidasic colorimetric analysis of S-acetyl-coenzyme-A (Wako Pure ChemicalIndustries, Osaka, Japan).Adopt colorimetric analysis (Triglyceride Procedure 336, Sigma Diagnostics) to measure plasma triglyceride.Nearest data show that also long-chain S-acetyl-coenzyme-A ester can be used as the labelling of lipid metabolism and insulin sensitivity in rat and people's the muscle (see Bronwyn etc., 2000, U.S. physiology magazine 279:E544-E560).Can measure by solvent extraction long-chain coenzyme A, PHASE SEPARATION from tissue with by reversed phase high efficiency liquid chromatography purification and organize long-chain coenzyme A (as derive from muscle dissect) (see as Bronwyn etc., 2000, U.S. physiology magazine 279:E544-E560).In addition, can also adopt the new method that is used to measure the intramuscular content of triglyceride.These methods comprise non-invasive method, (see as Kelley etc., 1991, clinical nutrition magazine (J.Clin.Nutr.) 54:509-515 as computed tomography art and nuclear resonance spectroscopy; Krssak etc., 1999, diabetologist 42:113-116; Jacob etc., 1999, diabetes 48:1113-1119).
In some embodiments of the invention, the CGRP receptor stimulating agent is applied to mammal, measures the vasodilation in this mammal.CGRP-1 and CGRP-2 are that effective vasodilation thing (is seen as Muff etc., 1995, European endocrinology magazine 133:17-20; Nuki etc., the same).But, suppose that CGRP-1 (with other agonist of high-affinity CGRP receptor) can partly distinguish vasorelaxation action with it at least to the influence that the skeletal muscle lipid decompose to stimulate, as activating high-affinity receptor and do not activate (or Min. ground activates) metabolism amylopectin receptor by the reagent of using one type, prescription or dosage.In one embodiment of the invention, this reagent is applied to mammal, measures the vasodilation of this animal.Can adopt any suitable analysis to detect vasodilation (opinion) as Nuki etc., 1993, biochemistry biophysical studies communication 196:245-51.In the mankind, can monitor vasodilation by measuring systolic pressure and mean arterial blood pressure.
In some embodiments of the invention, the CGRP receptor stimulating agent is applied to mammal, measures blood-glucose and generates and/or lactate level.As noted above, can realize that the lipid of high-affinity receptor mediation decomposes, and can not produce by the receptor-mediated not expectation function of metabolism amylopectin (raising) as blood glucose levels.
In some embodiments of the invention, the CGRP receptor stimulating agent is applied to mammal, and the symptom of the disease relevant with the muscle accumulation of lipid slows down in the monitoring mammal.In some embodiments of the invention, whether the monitoring disease process long enough time reduces with the order of severity, sign or the development of determining this symptom.In some embodiments of the invention, the CGRP receptor stimulating agent is applied to mammal (stimulating the mammal of lipid decomposition as needs), monitors one or more diseases or the physiological status of this animal.For example, in some embodiments of the invention, the CGRP receptor stimulating agent is applied to mammal, monitors the influence to insulin resistant of using of agonist in this mammal.Can evaluate insulin resistant in the animal by any the whole bag of tricks known in the art.For example, in the mankind, can adopt oral glucose tolerance test or OGTT (see as Bergman etc., 1985, endocrinology is looked back 6:45-86) to monitor insulin resistant.In a word, 75 grams carried out 2 hours, and the OGTT level is considered to insulin resistant greater than the individuality of 140mg/dl, and the individuality that is lower than the 120mg/dl level is considered to normal.(see as Reaven etc., 1979, diabetologist 16:17-24) monitors insulin resistant can also to adopt the blood-glucose of stable state to measure.The SSPG meansigma methods is considered to insulin resistant usually greater than the individuality of 180mg/dl; And the SSPG meansigma methods is considered to normal less than the individuality of 150mg/dl.Additive method comprises measures HbAlc and with labelling (del Prato, 1999, the medicine 58: appendix 1:3-6 of C-peptide as insulin resistant; Ferranini etc., 1998, hypertension 16:895-906).
Any above-mentioned monitoring work can merge to be carried out; Therefore, as in one embodiment, in the mammal of having used agonist, monitor vasodilation and insulin resistant simultaneously.In one embodiment, monitoring symptom (as insulin sensitivity/resistance) or use result's (decomposing) of agonist as lipid, use before the reagent (as generating baseline) and/or using after one or more time points (maybe, using the back) at one or many when being when repeatedly using measure.
In one embodiment of the invention, agonist is applied to the individuality that is diagnosed as insulin resistant.In one embodiment, this individuality is diagnosed as suffers from insulin resistant, but is not diagnosed as diabetes (as suffering from type 2 diabetes mellitus).
Instruct as this paper, lipid decomposition and vasodilative activity as open CGRP (or other CGRP receptor stimulating agents) can partly be distinguished at least, concerning experienced practitioner, obviously can be by result's (lipid decomposes enhancing, insulin sensitivity rising etc.) of monitoring vasodilation and expectation, give a certain amount of CGRP of administration or other agonist, be enough to stimulate lipid to decompose (thereby with reduce the muscle accumulation of lipid) and do not cause and only Min. ground strengthen vasodilation.
F. screening technique
Whether in one aspect, the invention provides a kind of method, be that the CGRP receptor stimulating agent determines whether this reagent is used in mammal or mammalian cell (as Skeletal Muscle Cell) moderate stimulation lipid decomposes by determining a kind of reagent.In one embodiment, determine the preferred reagent that activates high-affinity receptor (with respect to metabolism amylopectin receptor).As noted above, this reagent can be used for pharmaceutical compositions and the treatment that is used for disease and symptom.As described below, can comprise the EC of comparison agents influence by the definite preferred stimulation of variety of way to high-affinity receptor by the receptor-mediated reaction of metabolism amylopectin
50(as influence) and the EC of agents influence by the receptor-mediated reaction of high-affinity CGRP to carbohydrate metabolism
50(free fatty acid increases in skeletal muscle tissue or the Skeletal Muscle Cell).
Should be appreciated that and described hereinly determine and screening technique can be used to screen individualized compound or a large amount of chemical compound with treatment useful activity.This colony is at least 3 kinds of reagent, is more typically at least 25 kinds, is more typically at least 50 kinds, at least 100 kinds and comprise the very many reagent of screening (＞1000) usually.Screening is high-throughout and/or comprises order or the analysis carried out simultaneously (after being included in beginning, but finishing before this analysis carries out other reagent the analysis of initial a kind of reagent).
Any suitable analysis can be used to determine preferred the activation, includes, but is not limited to the EC that stimulates lipid to decompose by high-affinity receptor according to reagent
50(as reducing by tissue triglycerides or free fatty rising mensuration) and the EC of this reagent by the decomposition of metabolism amylopectin receptor for stimulating lipid
50Relatively come to determine or analyze determine (to see as II (B) and embodiment) with described herein other.Can recognize that these analyses can be used to screen plurality of reagents, as, library of compounds is as passing through the high flux mode.Can expect, by high-affinity CGRP receptor and the not instruction of same-action that this paper exists, diverse ways can be used to determine chemical compound, compositions, dosage and the prescription of or stimulation high-affinity CGRP receptor preferably exciting with respect to metabolism amylopectin receptor.Obviously these methods can also be used to screen the reagent that is used for the treatment of insulin resistant and other symptoms.
Screening analysis of the present invention can be adopted, and is not limited to, and animal, vitro tissue or organ cultures (as isolating skeletal muscle), cell or cell line are carried out, as, determine whether this chemical compound can be used for reducing the lipid content in animal, the cell or tissue.In addition, this agonist is applied to isolated cells system.See as, embodiment hereinafter.
In one embodiment, the invention provides and determine whether a kind of reagent can be used for the method for decomposing at mammal moderate stimulation lipid, determine at least a and be generally a large amount of reagent, itself and high-affinity CGRP receptors bind (as employing CGRP-1 substitutability analysis) by (i); (ii) select preferably to stimulate the reagent of high-affinity CGRP receptor with respect to metabolism amylopectin receptor.In related embodiment, the invention provides and determine whether a kind of reagent is used for the method for decomposing at mammal moderate stimulation lipid, determine at least a and common a large amount of reagent by (i), it decomposes at the moderate stimulation lipid of organizing of expressing high-affinity CGRP receptor; (ii) from (i), select preferably to stimulate the reagent of high-affinity CGRP receptor with respect to metabolism amylopectin receptor.In one embodiment, this method comprises (i) at least a and common a large amount of reagent, itself and high-affinity CGRP receptors bind; (ii) from (i), select preferably to stimulate the reagent of high-affinity receptor with respect to metabolism amylopectin receptor.In one embodiment, this tissue is skeletal muscle (as deriving from rat), and in one embodiment, this method comprises (i) at least a and common a large amount of reagent, itself and high-affinity CGRP receptors bind; (ii) select a kind of reagent from (i), its influence is by the EC of the receptor-mediated reaction of metabolism amylopectin
50Be higher than the EC of this agents influence by the receptor-mediated reaction of high-affinity CGRP
50In one embodiment, this tissue is skeletal muscle (as deriving from rat).
In another embodiment, the invention provides according to the EC of a kind of agents influence by metabolism amylopectin receptor and the receptor-mediated reaction of high-affinity CGRP
50, this reaction is described as mentioned and is generally the lipid decomposition, determines whether this reagent is used in the method that mammal moderate stimulation lipid decomposes.In one embodiment, this agents influence is by the EC of the receptor-mediated reaction of metabolism amylopectin
50Than the EC of this agents influence by the reaction of high-affinity receptor mediation
50High about 10 times, at least about 100 times, at least about 1000 times and preferably at least about 10,000 times, at least about 100,000 times or at least about 1,000,000 times.
In one embodiment, musculus soleus (as the musculus soleus that exsomatizes) is used to determine the lipid degrading activity of chemical compound.(the lipid degrading activity of Shi Yonging is meant the ability that a kind of reagent decomposes at cell or tissue moderate stimulation lipid in this article).Musculus soleus can be from the animal of high lipid food or normal diet of feeding, the dose-response curve of preparation chemical compound, wherein generate the analysis reflection (as measuring muscle free fatty and content of triglyceride) of the process that (i) take place by high-affinity CGRP receptor and metabolism amylopectin receptor and the process reflection that (ii) takes place by metabolism amylopectin receptor (when having the maximal stimulus insulin to the muscle glycogen content with [
14C] conversion of glucose is the mensuration of glycogen).From dose-response curve, determine EC
50Value is (as relative EC
50Value).Can from (i) and result (ii), select the dosage of chemical compound.Especially meaningfully in (i) and not in (ii), do not produce the compound concentrations of metabolic effect.In the comparison of different chemical compounds, this concentration will be the measured value of chemical compound relative potency.For example, according to its EC
50Value, if a kind of chemical compound under low relative concentration at (i) and not generation effect in (ii), this chemical compound will be considered to have the relative potency that is higher than other chemical compounds.
In a related aspect, determine the preferred reagent that activates high-affinity CGRP receptor (with respect to metabolism amylopectin receptor), comprise the different bonded step of detection high-affinity CGRP receptor (as skeletal muscle CGRP receptor) and metabolism amylopectin receptor (as muscle metabolism amylopectin receptor).Adopt different several different methods to carry out combination research expediently in conjunction with test by radioligand.For example, detectable with suitable radiation probe (as
125I and
3H) carry out radio-labeled, when existing and lacking excessive unlabelled part, cultivate with the prepared product that contains high-affinity CGRP receptor (as the skeleton sarolemma).Make up saturation curve then, obtain to be used for the incorporating parametric K of specific bond
d(concentration of the detectable that semi-saturation is required).In another embodiment, detectable can replace by the competitiveness of radiolabeled CGRP part to determine to the parameter of the specific bond of high-affinity CGRP receptor.In this case, the unmarked detectable that exists content to raise, radiolabeled CGRP cultivates with the prepared product that contains high-affinity CGRP receptor (as the skeleton sarolemma).The figure of the concentration of the detectable of drawing combined CGRP part and not being labeled will obtain sigmoid curve, therefrom obtain the inhibition constant K of detectable
iIt is well known in the art carrying out the method for binding analysis and the method for analytical reactions and data.See as
Www.graphpad.com/prism/And " classification of RBI receptor and signal conduction handbook, K.J.Watling, J.W.Kebebian, J.L.Neumeyer edits, Research BiochemicalsInternational, Natick, Mass., 1995, and list of references.The method of analyzing can be at " pharmacology analysis of drug receptor effect " of T.Kenakin, the 2nd edition, Raven Press, New York, 1993 and list of references in find.
In one aspect of the invention, high-affinity CGRP receptor stimulating agent (maybe may be the test compounds of agonist) is applied to animal, determines in screening is analyzed whether this chemical compound can be used for reducing the lipid content in the animal (as animal tissue).For example, this agonist can be applied to inhuman animal, and this animal is as the human diseases relevant with the muscle accumulation of lipid or the model of symptom.The example of this test model comprises the animal model as insulin resistant, as ob/ob (Kreutter, 1991, diabetes 40 (appendix 1): 159A (summary); Bretherton, endocrinology 129 (appendix 1): 91A (summary)), db/db (Kreutter, diabetes 40 (appendix 1): 159A (summary)); The yellow kind that can survive (obere-diabetic viable yellow) (Gill with obesity-diabetes in the mice, 1991 life sciences 48:703-710), and LA/N-cp (Huang, 1992 hypertension 19 (appendix 1): 101-109 (Figure 14), SHR/N-cp (Dunning, 1991 diabetes 40 (appendix 1)), fat Zucker (Kotanyi L 1992 diabetes 41:685-680), obesity-diabetes Zucker kind (Geduolin with rat, 1991 biochemistry biophysical studies communication 180:782-789), and by using glucocorticoid (Jamal H, 1990, endocrinology 126:425-429), golden yellow thioglucose (gold thioglucose) (Tokuyama, or abdomen center hypothalamus damage (Tokuyama, 1992 diabetes study clinical practices (the Diabetes Res Clin Pract) 15:23-29) animal that causes insulin resistant 1991 endocrinology 128:2739-2744); Tokuyama, 1991 endocrinology 128:2739-2744) except lipid content (performance of FFA), can estimate other effects such as blood-glucose and lactate level, the vasodilation degree determines can not cause the not chemical compound of expected result.Can expect that come the information of self-organizing and cell analysis can be used to determine compound concentrations, it is the most useful for carrying out extra-organismal dose response research.Whole animal screening study also can be used for assessing compound or dosage.Example operation and Ji-Ming etc. 2000, and it is similar to describe the method that is used for amylopectin among the U.S. physiology endocrine metabolism magazine 280:E562-569.Be appreciated that experimental animal is condemned to death in analysis embodiment more described herein in process of the test or afterwards.
When determining the EC of reagent (or the mixture of test agent, as in some high flux screening modes) for high-affinity CGRP receptor and/or metabolism amylopectin receptor
50The time, can adopt various analytical methods.For example, for the EC of specific chemical compound, receptor and effect
50, can determine for the dose-effect curve that fatty acid discharges in conjunction with the influence and the generation reagent concentration of high-affinity receptor CGRP-1 by analyzing various concentration parts.In one embodiment, when being high-affinity CGRP receptor, various concentration determination reagent are cultivated under the condition that keeps tissue survival with stripped musculus soleus cutlet.Behind specific cultivation stage, extract triglyceride and free fatty, quantize, with respect to the logarithm value graphing of the concentration of detectable.With sigmoid curve and data fitting, obtain EC from 50% of maximal stimulus required detectable concentration
50Value.Describe among the dose response data embodiment hereinafter of instance analysis and acquisition, see embodiment 4.In a word, the CGRP of certain limit concentration is added to (as skeletal muscle) in the sample that contains a large amount of receptors.By drawing CGRP concentration for the figure of CGRP in conjunction with effect (raising) indicant, the bonded EC of CGRP as the free-fat acid concentration
50Can be confirmed as causing half CGRP concentration of the maximal stimulus of detected effect (generating) as free fatty.
In one embodiment, analyze a kind of reagent (or a large amount of reagent, for example, collect (in pools) or high flux screening mode as described below) (a) analyze and determine that this reagent passes through the EC that high-affinity CGRP receptor (as skeletal muscle CGRP receptor) stimulates lipid to decompose
50, i.e. " EC
50-CGRP-R" and the EC that (b) analyze to determine stimulates lipid to decompose
50, i.e. " EC
50-amylopectin-R".Compare these two kinds of EC
50, have EC greater than about 10
50-amylopectin-R/ EC
50-CGRP-RThe reagent of ratio is considered to have the EC of the high-affinity receptor significantly lower than amylopectin receptor
50Preferred EC
50-amylopectin-R/ EC
50-CGRP-RThan being at least about 10, usually at least about greater than about 10
2, greater than 5 * 10
2, greater than about 10
3, greater than 5 * 10
3, greater than about 10
4, greater than about 10
5, greater than about 10
6, greater than about 10
7, or greater than about 10
8
Can screen with various types of compounds and compositions.Suitable test agent comprises natural and synthetic chemical compound or compositions.Suitable detectable comprises polypeptide (comprising albumen and small peptide, as described herein the CGRP related peptides), carbohydrate such as oligosaccharide and polysaccharide; Polynucleotide; Lipid and phospholipid; Fatty acid; Steroid; Dipeptides, amino acid analogue, organic molecule are as micromolecule (as molecular weight less than 1000).The chemical compound that detects can be various chemical specieses, includes but not limited to heterocyclic chemical compound, carbocyclic compound, lactams, polycarbamate.In one embodiment, reagent is determined by the SCREENED COMPOUND library.The foundation in synthetic molecules library and screening can adopt the technology of knowing in the combinatorial chemistry to carry out, and for example see van Breemen, 1997 analytical chemistry 69:2159-64; Lam, 1997, cancer therapy drug research 12:145-67; Gold, 1995, journal of biological chemistry 270:13581-84).In addition, the active improved chemical compound that comes in handy in a large number can screened from as the extract of raw-material natural products.The source of this extract can be from many species, as fungus, actinomycetes, algae, insecticide, protozoacide, plant and antibacterial.Analyzing these then has active extract and comes the isolating active molecule.See as, Turner, 1996, national pharmacy (Ethnopharmacol) 51:3943; Suh, 1995, anticancer research (Anticancer Res) 15:233-39.Many other test agent and libraries are that this area is known.In certain embodiments, at least 100 kinds of different test compounds and reagent are contained in the test agent library.
In one aspect, the invention provides lipid degrading activity by more a kind of reagent and the lipid degrading activity of CGRP and determine whether this reagent is used in the method that mammal moderate stimulation lipid decomposes.According to this analytical method, (in skeletal muscle or liver) reagent identical with CGRP or that stimulate lipid to decompose (comparing) better on identical molal quantity basis is considered to be used in the decomposition of mammal moderate stimulation lipid in cell or tissue, is the candidate compound of animal and human's class treatment.Can in parallel analysis, (carry out) comparing step simultaneously.In addition, the standard value of lipid degrading activity (as every mole of FFA that reagent generated in the unit interval under given conditions) and precedence record (as medium at computer-readable medium) relatively.Also can compare indirectly: for example, compd A and CGRP be the lipid degrading activity relatively, and the lipid degrading activity of the lipid degrading activity of test agent and compd A compares (thereby comparing with CGRP indirectly).In one aspect, by the lipid degrading activity basis as a comparison of the CGRP of high-affinity receptor mediation.For example, in one embodiment, carry out the active comparison of CGRP lipid degrading activity under the 300pM concentration being lower than.For example, in one embodiment, under the concentration that the lipid that can substantially not stimulate metabolism amylopectin receptor decomposes, carry out the comparison of CGRP lipid degrading activity.On the other hand, by high-affinity receptor and metabolism amylopectin receptor or by the metabolism amylopectin receptor lipid degrading activity basis as a comparison of the CGRP of mediation separately.In one embodiment, any form A GRP that decomposes at skeletal muscle moderate stimulation lipid can use; In relevant embodiment (is obvious according to aforementioned content), the CGRP form is to stimulate lipid to decompose by high-affinity receptor.The canonic form of CGRP is but is not limited to rat and people's CGRP-1.
G. suppress the method that lipid decomposes
In yet another aspect, the invention provides the method for coming in skeletal muscle, to suppress the lipid decomposition by the antagonist of using high-affinity and/or metabolism receptor.Under test and clinical condition, it is useful suppressing the lipid decomposition in tissue.For example, the chronic administration antagonist may cause the compensation metabolic process in muscle and liver, causes that the lipid utilization strengthens (as raising beta oxidation) and the muscle lipid that causes expecting reduces.The reagent that suppresses the lipid decomposition also can be used for drug screening analysis (decomposing the forward and the retroregulation of actuator as determining lipid).Antagonist can be any chemical compound that suppresses by the lipid decomposition of a kind of reagent such as CGRP and amylopectin stimulation.Antagonist may suppress in conjunction with or competition to the combination of high-affinity CGRP receptor or low affinity metabolism amylopectin receptor (see as hereinafter embodiment 4).Antagonist can be a native compound, as a kind of protein, peptide, polynucleotide or little effector molecule or by synthetic preparation.In one embodiment, antagonist is
8,37Amylopectin or
8,37CGRP.
8,37CGRP and
8,37Amylopectin refers to the variant of CGRP and amylopectin respectively, and wherein preceding 7 aminoacid of C-end are removed.(see Cooper, 2001, " endocrine pancreas and to the metabolic regulation and control " II (7) in physiology's handbook: hormonal system, Jefferson, L.S., Cherrington, A.D. edits, the Oxford University Press).Can also use non-peptide CGRP antagonist.Comprise BIBN4096BS (see Wu etc., 2002, BichemSoc.Trans.30:468-73), " chemical compound-1 " and " chemical compound-2 " (see Mallee etc., 2002, journal of biological chemistry 277:4294-98).
This antagonist is applied to needs with the treatment effective dose and suppresses in the mammal of lipid decomposition.This receptor antagonist can combine and be applied to animal with pharmaceutically acceptable excipient.The mode of using is similar to using of above-described receptor stimulating agent.
Materials and methods
Present embodiment is described in the materials and methods that uses in the described test of embodiment 2-7.
Material unless otherwise noted, all chemical drugss are AGs or more high-grade.Pentobarbital sodium (nembutal) is available from Virbac Laboratories Ltd.AC128, AC128-(8-37), rat CGRP1 and human CGRP 1-(8-37) (seeing accompanying drawing 1) be available from Bachem, Switzerland.NaHCO
3, CaCl
2, D-glucose, NaCl, KH
2PO
4, chloroform, methanol, HCl, NH
4OH, ethanol and Triton X-100 are available from BDH; MgSO
47H
2O is available from Sigma; With KCl available from Riedel-de Haen.Politef post (16ml), politef frit (teflon frits) and vacuum elution instrument (Vac Elutapparatus) are available from Alltech.Bonded silicon 40 μ of aminopropanol (key elution (Bond Elut)) column packing is available from Phenomenex; Hexane and diethyl ether are available from Labscan.Heptane available from Water Associates Inc and boron trifluoride-methanol complex (BF3 is dissolved in the methanol) available from Aldrich Chemical CompanyInc.Butanols modification hydroxy toluene and all fatty acid methyl ester reference materials are available from Sigma.GLC post DB225 (30m * 25mm I.D.) derives from J﹠amp; W Scientific.Three-N-octyl amine is available from Acros Organics; Trichloroacetic acid is available from Ajax Chemicals Ltd; With glycerol available from Scharlau.Free fatty, Half-microtest are available from Boehringer Mannheim.The lipid enzyme releasing agent powder that is used for glycerol detection and triglyceride detection is available from Pointe Scientific Inc.
The animal model rat all operations of normally feeding obtains University of Auckland animal Ethics Committee approval.The heavy 200-250g of male Wistar rat (age=50 day) is in illumination/dark cycle of lasting 12h/12h, and random foraging standard rat material (Diet 86, NRM, Auckland) and water.Standard food contains 5% lipid (w/w), mainly contains Adeps Bovis seu Bubali.Analyze the triglyceride of the representative sample of this food, total and free content of fatty acid, be shown in Table 3.The peritoneal injection pentobarbital sodium comes anesthetized rat (45mg/kg body weight), takes off neck then and puts to death.
Table 3
The fatty acid composition of standard rat material
Free fatty and TL sample adopt gas chromatographic analysis, and mass spectrum is passed through in being confirmed to be that individual fatty acid is identified
Instrument is analyzed, and with the free fatty and the total fatty acids compositions of the rat material that settles the standard, value is that average is poor (right
Every bit n=7).
Fatty acid free fatty fatty acid
(μmol/g)???????????(μmol/g)
Tetradecanoic acid C14:0 10.52 ± 2.44 12.54 ± 3.26
Pentadecanoic acid C15:0 7.93 ± 1.44 8.90 ± 0.324
Hexadecanoic acid C16:0 508 ± 97.8 610 ± 21.8
Hexadecenoic acid (n=9) C16:1 7.10 ± 1.30 8.91 ± 0.469
Hexadecenoic acid (n=7) C16:1 33.0 ± 6.14 39.1 ± 1.48
Heptadecanoic acid C17:0 9.57 ± 2.12 11.6 ± 0.385
Octadecanoid acid C18:0 173 ± 40.2 196 ± 4.74
Oleic acid (n=9) C18:1 22.8 ± 3.43 1293 ± 70.4
Octadecenoic acid (n=7) C18:1 1.99 ± 0.424 37.7 ± 0.892
Octadecadienoic acid C18:2 642 ± 62.8 1081 ± 41.4
Jeceric acid C18:3 96.9 ± 17.9 166 ± 6.46
Arachic acid C20:0 10.9 ± 2.08 12.2 ± 0.439
Eicosatetraenoic acid C20:4 2.13 ± 0.311 7.28 ± 0.288
Eicosapentaenoic acid C20:5 25.7 ± 4.57 46.6 ± 1.47
Docosatetratenoic acid C22:4 1.41 ± 0.312 1.69 ± 0.0711
Docosahexenoic acid C22:6 4.12 ± 0.708 9.61 ± 0.354
Rat array male Wistar rat (n=21/ food) free choice feeding that high lipid is fed, contain the high lipid food of standard rat material in putting to death (n=50d), feeding, wherein add 40% (w/w) Adeps Sus domestica, Semen Maydis oil and olive oil from wean (n=21d).Triacylglycerol in the Adeps Sus domestica mainly contains satisfied fatty acid; Triacylglycerol in the Semen Maydis oil is mainly monounsaturated fatty acid; And the triacylglycerol in the olive oil is mainly polyunsaturated fatty acid, sees Table 4.
Table 4
Be used for preparing the free fatty composition of the lipid of high lipid food
The free fatty composition of Adeps Sus domestica, Semen Maydis oil and olive oil.Numerical value is the % of total amount.
Fatty acid Adeps Sus domestica Semen Maydis oil olive oil
C16:00??????????????26.9??????????????5.0?????????????????15.2
C16:1???????????????3.5???????????????--??????????????????1.7
C18:0???????????????51.8??????????????1.7?????????????????2.7
C18:1(n-9)??????????3.5???????????????10.7????????????????66.7
C18:1(n-7)??????????--????????????????3.9?????????????????3.0
C18:2???????????????14.3??????????????57.2????????????????10.5
C18:3???????????????--????????????????21.1????????????????--
The separation of musculus soleus tissue and cultivation are soaked and the solution of cultivation is Krebs-Henseleit (KHB), contain 118.5mM NaCl, 4.75mM KCl, 1.18mM MgSO
4.7H
2O and KH
2PO
4, 24.8mM NaHCO
3, 2.54mM CaCl
2With 10mM D-glucose.In this solution, continue logical people 95%O
2/ 5%CO
2With independent KHB or contain CGRP, amylopectin, norepinephrine, insulin or the CGRP of various concentration aptly and the antagonist of amylopectin is cultivated.The musculus soleus of excision both legs is immersed among the KHB; Muscle is divided into two equivalent parts, transfers in the culture bottle.After cultivating end, rapidly muscle is chilled in the liquid nitrogen, removes connective tissue and all visible lipids then.Strip of muscle is preserved up to analysis down at-80 ℃.
The liquid extraction of fatty acid and chromatographic isolation adopt method (Gorski etc., 1998, molecule and the cellular biochemistry 178:113-118 of improved Bligh and Dyer; 1.Bligh etc., 1959, Canadian biochemistry physiology magazine 37:911-917) from strip of muscle, extract TL.Muscle homogenize in the 0.4ml deionized water (Soniprep 150).(Bligh and Dyer, the same) as described, all solvent volume change according to the moisture in the muscle (muscle weight).The methanol and the 1 volume chloroform that two volumes are contained 0.005% butanols modification hydroxy toluene add in the muscle suspension.Stir homogenate 2 minutes with maximal rate, with 2, centrifugal 4 minutes of 500g.Remove supernatant, extract precipitate again with 2 volumes methanol, 1 volume chloroform and 0.8 volume 0.2N HCl.Stirred residue 2 minutes, then with 3, centrifugal 3 minutes of 500g.After centrifugal, blended supernatant is with the dilution of two volume milli Q water and chloroform, and by with 3,500g carried out PHASE SEPARATION in centrifugal 4 minutes.Take out lower chloroform phase, drip 0.2N methanolizing NH
4OH neutralizes, evaporation drying in liquid nitrogen steam.Sample is preserved standby down at-80 ℃.
Aminopropanol phase (250mg) is added upper strata filled polytetrafluoroethylene frit fills in the bonding 16ml politef post mutually with the bottom.Post is put into vacuum elution instrument, with 2ml hexane eluting twice (Prasad etc., 1988, chromatograph magazine (J Chromato) 428:221-228; Kaluzny etc., 1985, lipid research magazine (J LipidRes) 26:135-140).The lipid sample of doing is dissolved in the 0.150ml chloroform, and adds in the post under normal pressure.After absorption, (2: 1, v/v) eluting neutral lipid was blended in 2% acetic acid eluting free fatty in the diethyl ether with 4ml with 4ml chloroform-2-propanol.The solvent that contains free fatty is at N
2Dry in the steam.
Vapour liquid chromatography with the boron trifluoride-methanol method handle exsiccant fatty acids residues derive methyl ester (Prasad, above).The boron trifluoride (BF3) that 1ml is dissolved in the methanol adds in each sample, and specimen bottle heated 5 minutes down at 70 ℃, acutely rocked reheat 10 minutes.In case sample returns to room temperature, add 0.5ml milliQ water, rock specimen bottle, add the 0.1ml heptane, rock specimen bottle again.The heptane layer of taking out 0.05ml top is used for GLC and analyzes.The model that the DB-225 post is housed is that the gas chromatograph of HP5890 GC Plus Series (Hewlett-Packard) is used for the separation of fatty acids methyl ester, and the model that model is housed is HP5973 MS (Hewlette-Packard) is that the GC/MS of HP5890 GC is used to confirm determining various free fatties.Heating schedule then in the end stopped 10 minutes under the temperature for to be elevated to 210 ℃ of final temperatures with 3 ℃/min from 80 ℃ of initial temperatures linearly.Tissue fat acid quantizes to depend on the retention time of fatty acid methyl ester reference material and the reaction factor of correlation theory.Determine free fatty according to reference material and GC/MS chromatograph.
By free fatty and the triacylglycerol concentration separation of free fatty acids from TL in the definite tissue of enzymatic analysis, at N
2Evaporation drying in the steam, and be kept under-80 ℃ up to being used for analysis.Sample is dissolved in the 50 μ l hot ethanols (35-40 ℃), when ethanol returns to room temperature, adds Triton X-100 solution (6%w/v).Agitating solution 30min adds Triton solution to 0.825ml.Quantize free fatty with the Palmic acid reference material by being adjusted to the business method (Cobal Mira, Roche Diagnostics) that is fit to microanalysis.In TL fraction (Pointe Scientific), quantize the triacylglycerol level with the glycerol reference material.
The refrigerated muscular tissue of glycerol in the enzymatic analysis tissue is ground, and is weighed in the 2ml centrifuge tube.In each sample, add 0.5ml10% (w/v) trichloroacetic acid, homogenize 4 minutes in ice cube then.Centrifugal and rock homogenate repeatedly, after placing 2 minutes on the ice cube, with 10, the centrifugal 5min of 000rpm.0.4ml supernatant is transferred in the new test tube, adds 0.5ml three-N-octyl amine/Freon 113 (1: 2.03v/v).Mixed solution, and on ice cube, placed 5 minutes.Measure the pH of upper aqueous layer, sample is mixed and centrifugal up to obtaining neutral pH.Take out the 0.2ml upper strata and analyze glycerol content.
All results of statistical analysis are expressed as average ± S.E.M..For of the influence of average hormone, carry out variable single factor analysis (ANOVA) to lipid metabolism.Dunnett post hoc check is used for the difference between analyzing and processing group and the corresponding control value.All statistical analysiss carry out with the Macintosh Graph Pad Prism program of Power PC version.The numerical value of P＜0.05 thinks to have statistical significance.
Amylopectin, CGRP, norepinephrine and insulin are to lipid in the skeletal muscle that derives from the rat of normally feeding
Metabolic influence.
Present embodiment is described the stimulation to the lipid metabolism level in the isolated rat musculus soleus tissue that is caused by all ingredients.Muscular tissue is handled with amylopectin, CGRP, norepinephrine or insulin, and the level and the control level of free fatty, glycerol and triglyceride compare.
Amylopectin, CGRP, insulin and norepinephrine are presented among the accompanying drawing 2A the influence of the free fatty acid content in the musculus soleus that exsomatizes.Free fatty acid content in the musculus soleus of handling with amylopectin, CGRP and norepinephrine is compared with the free fatty acid content of contrast muscle has significant difference.(295 ± 27nmol/g is than 78 ± 21 with norepinephrine, P＜0.01) and amylopectin (216 ± 22nmol/g is than 78 ± 21, P＜0.01) compare, cultivating the maximum cause the intramuscular free fatty with CGRP increases (325 ± 56nmol/g than 78 ± 21, P＜0.01).Compare with control tissue, usually cultivate the concentration (67 ± 9nmol/g is than 78 ± 21) that musculus soleus can not change the intramuscular free fatty with the maximal stimulus islets of langerhans.
Shown in accompanying drawing 2B, amylopectin, CGRP and norepinephrine cause that the intramuscular glycerol content descends.Behind amylopectin, CGRP and norepinephrine cultivation 1h, glycerol content is respectively that (0.03 ± 0.004nmol/g is than 0.07 ± 0.007, P＜0.01), (0.04 ± 0.008nmol/g is than 0.07 ± 0.007, P＜0.01) and (0.04 ± 0.005nmol/g than 0.07 ± 0.007, P＜0.01).
Accompanying drawing 2C is illustrated in and cultivates the triacylglycerol content in the musculus soleus behind the 60min.Handle reduction triacylglycerol content (0.81 ± 0.09nmol/g is than 1.17 ± 0.15) with norepinephrine.Handle the trend of rising (1.7 ± 0.26nmol/g (amylopectin), 1.9 ± 0.23nmol/g (CGRP) are than 1.17 ± 0.15 (contrasts)) in addition of muscle triacylglycerol with amylopectin and CGRP.But do not have marked difference between these results and the control value.
Detect (each handles n=7) from three kinds of hormone-treated culture mediums with identical enzyme substrate method.Do not detect and be discharged into end free fatty or glycerol (result does not show) in the medium.
Hormone is to the influence of the various free fatty acid contents in the skeletal muscle
Present embodiment is described the quantification of the various free fatties that discharge in the skeletal muscle that response CGRP, amylopectin or norepinephrine are handled.All programs adopt gas chromatogram to quantize various free fatties, determine to identify by mass spectral analysis (GC-MS).Fatty acid level in all lipid fractions of table 5 is illustrated in static (resting) musculus soleus is determined by analyzing total lipid sample.Total baseline lipid sample is determined the static fatty acid composition in the musculus soleus by gas chromatographic analysis, determines discriminating to various fatty acids with mass spectral analysis.Numerical value is average ± S.E.M. (each point n=8).
Table 5
The fatty acid composition of musculus soleus
Fatty acid F A concentration (nmol/g wwt)
Tetradecanoic acid C14:0 0.78 ± 0.45
Pentadecanoic acid C15:0 0.1 ± 0.033
Hexadecanoic acid C16:0 8.05 ± 2.65
Hexadecenoic acid (n=9) C16:1 0.11 ± 0.05
Hexadecenoic acid (n=7) C16:1 0.1 ± 0.05
Heptadecanoic acid C17:0 0.133 ± 0.033
Octadecanoid acid C18:0 4.25 ± 0.733
Oleic acid (n=9) C18:1 4.9 ± 1.95
Octadecenoic acid (n=7) C18:1 1.25 ± 0.316
Octadecadienoic acid C18:2 5.5 ± 1.55
Jeceric acid C18:3 0.25 ± 0.1
Arachic acid C20:0 0.183 ± 0.13
Eicosatetraenoic acid C20:4 1.56 ± 0.316
Eicosapentaenoic acid C20:5 0.28 ± 0.083
Docosatetratenoic acid C22:4 0.083 ± 0.016
Docosahexenoic acid C22:6 0.816 ± 0.166
The release of the various free fatties that table 6 expression hormone causes.Show that C16:0 is the free fatty of the maximum that exists in the musculus soleus, still, cultivate when having hormone and can not cause that concentration raises.(0.013 ± 0.0011nmol/g is than 0.008 ± 0.0013 with amylopectin, P＜0.05), (0.013 ± 0.0011nmol/g is than 0.008 ± 0.0006 for CGRP, P＜0.01) and norepinephrine (0.013 ± 0.0011nmol/g than 0.007 ± 0.0007, P＜0.01) handle the concentration that reduces intramuscular C16:1 (n=9).
By HORMONE TREATMENT, concentration some long-chains, polyunsaturated fatty acids raises.(0.005 ± 0.001nmol/g is than 0.016 ± 0.002 with amylopectin, P＜0.01), (0.005 ± 0.001nmol/g is than 0.018 ± 0.002 for CGRP, P＜0.01) and norepinephrine (0.005 ± 0.001nmol/g than 0.015 ± 0.0007, P＜0.01) handle and improved intramuscular eicosatetraenoic acid (C20:4) concentration.Also improved docosahexenoic acid concentration with these three kinds of HORMONE TREATMENT: (0.01 ± 0.0008nmol/g is than 0.03 ± 0.004 for amylopectin, P＜0.01), (0.01 ± 0.0008nmol/g is than 0.022 ± 0.002 for CGRP, P＜0.01) and norepinephrine (0.01 ± 0.0008nmol/g than 0.022 ± 0.0015, P＜0.05).
Table 6
The release of the various free fatties that hormone causes
Analyze the release of the various free fatties that surpass 1h that cause by hormone in the musculus soleus with gas chromatograph, determine evaluation various free fatties by mass spectral analysis.Numerical value is nmol/g wwt, and is expressed as average ± S.E.M..(each n=16).* with the marked difference of comparing (P＜0.05).* and the marked difference (P＜0.01) of comparing.
Free-fat contrast amylopectin CGRP norepinephrine
Acid
Tetradecanoic acid C14:0 0.025 ± 0.002 0.026 ± 0.0028 0.028 ± 0.001 0.023 ± 0.001
Pentadecanoic acid C15:0 0.01 ± 0.00005 0.013 ± 0.002 0.01 ± 0.01 0.01 ± 0.0012
Hexadecanoic acid C16:0 0.17 ± 0.74 0.21 ± 0.0188 0.23 ± 0.01* 0.175 ± 0.011
Hexadecene C16:1 0.013 ± 0.0011 0.008 ± 0.0013*, 0.008 ± 0.0006* 0.007 ±
Acid (n=9) 0.0007**
Hexadecene C16:1 0.013 ± 0.002 0.015 ± 0.0025 0.022 ± 0.002* 0.015 ± 0.002
Acid (n=7)
Heptadecanoic acid C17:0 0.008 ± 0.0006 0.013 ± 0.0022*, 0.015 ± 0.0015* 0.01 ± 0.0007
Octadecanoid acid C18:0 0.09 ± 0.0035 0.0125 ± 0.0058**, 0.15 ± 0.01** 0.1 ± 0.006
Oleic acid (n=9) C18:1 0.06 ± 0.0051 0.076 ± 0.0082 0.09 ± 0.0075** 0.07 ± 0.0056
Vaccenic acid C18:1 0.008 ± 0.0007 0.015 ± 0.0015** 0.017 ± 0.015 ±
Acid (n=7) 0.0015** 0.0008**
18 carbon, two C18:2 0.035 ± 0.0045 0.058 ± 0.006**, 0.057 ± 0.006* 0.043 ± 0.003
Olefin(e) acid
18 carbon, three C18:3 0.001 ± 0.0007 0.01 ± 0.002* 0.005 ± 0.0013 0.007 ± 0.0025
Olefin(e) acid
Arachic acid C20:0 0.007 ± 0.001 0.008 ± 0.002 0.007 ± 0.0008 0.003 ± 0.001
20 carbon, four C20:4 0.005 ± 0.001 0.016 ± 0.002**, 0.018 ± 0.002** 0.015 ±
Olefin(e) acid 0.0007**
20 carbon, five C20:5 0.002 ± 0.0007 0.005 ± 0.0007** 0.003 ± 0.0005 0.002 ± 0.0005
Olefin(e) acid
22 carbon C22:4 0.002 ± 0.001 0.008 ± 0.003* 0.007 ± 0.002 0.007 ± 0.001
Tetraenoic acid
22 carbon C22:6 0.01 ± 0.0008 0.03 ± 0.004**, 0.022 ± 0.002** 0.022 ±
Acid 0.0015*
Table 7 expression intramuscular free fatty is with respect to the percentage ratio of total fatty acids level.This form has also shown the variation of the percentage ratio of obtainable free fatty after HORMONE TREATMENT.
Table 7
The release of the free fatty that hormone causes
The release of the free fatty that surpasses 1h that is caused by hormone in musculus soleus and total static fatty acid are relatively.Numeric representation is caused the ratio of the free fatty of release with respect to total static fatty acid level by HORMONE TREATMENT.
Fatty acid contrast amylopectin CGRP norepinephrine
C14:0?????????????3.2?????????3.4?????????????3.6?????????3.1
C15:0?????????????12.9????????15.4????????????12.9????????10.9
C16:0?????????????2.2?????????2.6?????????????2.9?????????2.2
C16:1(n-9)????????10.6????????7.2?????????????7.5?????????6.0
C16:1(n-7)????????12.6????????15.8????????????21.6????????14.3
C17:0?????????????6.3?????????10.7????????????10.7????????7.2
C18:0?????????????2.1?????????2.9?????????????3.6?????????2.4
C18:1(n-9)????????1.2?????????1.6?????????????1.8?????????1.4
C18:1(n-7)????????0.6?????????1.3?????????????1.4?????????1.2
C18:2?????????????0.6?????????1.1?????????????1.0?????????0.8
C18:3?????????????0.8?????????3.7?????????????2.1?????????2.5
C20:0?????????????3.7?????????4.5?????????????3.6?????????2.0
C20:4?????????????0.3?????????1.0?????????????1.1?????????0.9
C20:5?????????????0.5?????????1.4?????????????1.0?????????0.4
C22:4?????????????1.2?????????9.1?????????????8.1?????????7.5
C22:6?????????????1.3?????????3.3?????????????2.7?????????2.5
Free fatty acid levels and total fatty acid content in the table 3 expression standard rat material of front.
Amylopectin, CGRP and norepinephrine contain the musculus soleus triacylglycerol of the rat of the high lipid food of feeding
The influence of amount
The rat that high lipid is fed is the model of insulin resistant, is useful especially model (comparing with the animal of normally feeding) in the type 2 diabetes mellitus research.Top table 4 expression is used to prepare the lipid components of high lipid food.Table 8, the 9 and 10 expressions various free fatty acid contents in the musculus soleus of rat of one of these three kinds of high lipid foods of feeding.Accompanying drawing 16 shows that the rat of the high lipid food of feeding is an insulin resistant, determines from the insulin response of musculus soleus.In normal diet of feeding (■) or 51 days rat soleus muscle of high lipid food (), measure the dose-effect curve of insulin.After cultivating 2h, pass through D[with various concentration insulins
14C (U)] conversion of glucose is that inose was measured insulin response originally.Extract muscle glycogen then and analyze D[by the liquid scintillation spectrophotometer
14C (U)] concentration of glucose.Dose-response curve and the match of S shape dose response algorithm.The result is expressed as average ± SEM (each n=12-18 strip of muscle).Total sum of squares the analysis showed that extremely remarkable (P＜10 of difference between these two kinds of curves
-5).
Three kinds of hormones of accompanying drawing 6 expressions are to the influence of the content of triglyceride in the rat soleus muscle of one of the three kinds of high lipid foods of feeding.With compare accordingly, in the group of the food that contains 40% Adeps Sus domestica (accompanying drawing 6A) that adds of feeding, the remarkable decline that all three kinds of hormones cause content of triglyceride in the musculus soleus (in all cases, be that 0.75 ± 0.08 (CGRP), 0.71 ± 0.07 (amylopectin) and 0.69 ± 0.09 (norepinephrine) μ mol/g are than 1.14 ± 0.16 μ mol/g, p＜0.05).(0.48 ± 0.06 (CGRP) and 0.49 ± 0.07 (amylopectin) μ mol/g are than 0.89 ± 0.06 μ mol/g in all cases in the group of 40% Semen Maydis oil of feeding (accompanying drawing 6B), P＜0.05) or 40% olive oil (accompanying drawing 6C) (0.52 ± 0.08 (CGRP) and 0.44 ± 0.4 (amylopectin) μ mol/g are than 0.83 ± 0.1 μ mol/g in all cases, P＜0.05), amylopectin and CGRP cause remarkable reduction with only comparing.
Three kinds of hormones of accompanying drawing 7 expressions are to the influence of the free fatty acid content in the rat soleus muscle of one of the three kinds of high lipid foods of feeding.In the group of the food that contains 40% Adeps Sus domestica (accompanying drawing 7A) that adds of feeding, with compare accordingly, amylopectin and norepinephrine cause remarkable rising (102 ± 12 (amylopectins of free content of fatty acid in the musculus soleus, P＜0.01), 75 ± 7 (norepinephrine, P＜0.05) nmol/g is than 43 ± 8).CGRP has the free fatty acid content of causing trend of rising, and does not have marked difference (64 ± 7nmol/g is than 43 ± 8) between the control value.At 40% Semen Maydis oil of feeding (accompanying drawing 7B) (75 ± 9 (amylopectins, P＜0.05) and 99 ± 17 (CGRP, P＜0.01) nmol/g is than 35 ± 4nmol/g) or 40% olive oil (accompanying drawing 7C) (85 ± 12 (amylopectins, P＜0.01) and 55 ± 14 (CGRP, P＜0.05) nmol/g is than in 15 ± 3nmol/g) the group, and CGRP and amylopectin all cause the remarkable increase of musculus soleus free fatty acid content.
Use CGRP-1 and the effect of triglyceride and free fatty is taken place for animal that high lipid feeds by high-affinity CGRP1 site, this is by similar EC
50The dose-response value proves.Accompanying drawing 17A represents that CGRP-1 causes that the dosage of muscle NEFA content rely on to raise, and has the similar EC in observed high-affinity site in the muscle with normal feeding animals
50(0.7pM ± 0.4 is to 1.0; Average ± 95%C.I.).Accompanying drawing 17B represents that increasing relevant with NEFA concentration is that the dose dependent of total intramuscular content of triglyceride descends, and this is unobserved result in the musculus soleus of normal feeding animals.The EC that the intramuscular triglyceride reduces
50(0.25pM ± 0.03 is to 1.96; Average ± 95%C.I.) is observed EC in NEFA content raises
50Test error in determine.With respect to the muscle of normal feeding animals, the baseline content of triglyceride in the muscle of high lipid feeding animals significantly raises.In this test, the food 30 days that animal begins to feed and contains a large amount of saturated lipids (Adeps Sus domestica) from wean.
Table 8
The various free fatties that hormone causes in the rat soleus muscle of the food of the high Semen Maydis oil content of feeding discharge
The release gas chromatograph analysis of the various free fatties that surpass 1h that caused by hormone in the food rat soleus muscle of high Semen Maydis oil content of feeding is by the definite evaluation to various fatty acids of mass spectral analysis.Numerical value is nmol/g wwt, and is expressed as average ± S.E.M..(each n=16).* with the marked difference of comparing (P＜0.05).* and the marked difference (P＜0.01) of comparing.
Free-fat contrast amylopectin CGRP norepinephrine
Acid
Tetradecanoic acid C14:0 0.02 ± 0.001 0.04 ± 0.006**, 0.03 ± 0.02** 0.02 ± 0.002
Pentadecanoic acid C15:0 0.01 ± 0.001 0.05 ± 0.007**, 0.04 ± 0.03**, 0.04 ± 0.005**
Hexadecanoic acid C16:0 0.07 ± 0.006 0.12 ± 0.01 0.16 ± 0.03** 0.09 ± 0.01
Hexadecene C16:1 0.12 ± 0.007 0.19 ± 0.008**, 0.20 ± 0.008** 0.15 ± 0.01
Acid (n=9)
Hexadecene C16:1 0.005 ± 0.0005 0.01 ± 0.0008**, 0.01 ± 0.0008** 0.007 ± 0.0005
Acid (n=7)
Heptadecanoic acid C17:0 0.03 ± 0.006 0.02 ± 0.0009 0.02 ± 0.003 0.04 ± 0.006
Octadecanoid acid C18:0 0.09 ± 0.004 0.14 ± 0.008 0.39 ± 0.02 0.11 ± 0.008
Oleic acid C18:1 0.08 ± 0.008 0.13 ± 0.01* 0.16 ± 0.01 0.12 ± 0.02
Vaccenic acid C18:1 0.02 ± 0.002 0.02 ± 0.002 0.02 ± 0.004 0.02 ± 0.001
Acid
18 carbon, two C18:2 0.08 ± 0.005 0.18 ± 0.02**, 0.18 ± 0.008**, 0.14 ± 0.02*
Olefin(e) acid
18 carbon, three C18:3 0.07 ± 0.005 0.16 ± 0.02**, 0.17 ± 0.01**, 0.13 ± 0.01**
Olefin(e) acid
Arachic acid C20:0 0.03 ± 0.003 0.04 ± 0.002 0.03 ± 0.005 0.03 ± 0.002
20 carbon, four C20:4 0.02 ± 0.002 0.04 ± 0.002 0.02 ± 0.002 0.08 ± 0.009**
Olefin(e) acid
20 carbon, five C20:5 0.02 ± 0.002 0.05 ± 0.007**, 0.04 ± 0.001**, 0.05 ± 0.005**
Olefin(e) acid
22 carbon C22:4 0.02 ± 0.002 0.03 ± 0.003 0.04 ± 0.002**, 0.05 ± 0.004**
Tetraenoic acid
22 carbon C22:6 0.01 ± 0.0007 0.03 ± 0.002**, 0.03 ± 0.003**, 0.04 ± 0.003**
Acid
Table 9
The various free fatties that hormone causes in the rat soleus muscle of the food of the high content of olive oil of feeding discharge
The release gas chromatograph analysis of the various free fatties that surpass 1h that caused by hormone in the food rat soleus muscle of the high content of olive oil of feeding is by the definite evaluation to various fatty acids of mass spectral analysis.Numerical value is nmol/g wwt, and is expressed as average ± S.E.M..(each n=16).* with the marked difference of comparing (P＜0.05).* and the marked difference (P＜0.01) of comparing.
Free fatty contrast amylopectin CGRP norepinephrine
Tetradecanoic acid C14:0 0.004 ± 0.0003 0.01 ± 0.001**, 0.015 ± 0.0009** 0.005 ± 0.0002
Pentadecanoic acid C15:0 0.009 ± 0.0020 0.04 ± 0.005**, 0.08 ± 0.002** 0.01 ± 0.0004
Hexadecanoic acid C16:0 0.07 ± 0.02 0.18 ± 0.01 0.20 ± 0.006**, 0.19 ± 0.002*
Hexadecenoic acid C16:1 0.16 ± 0.03 0.29 ± 0.03**, 0.27 ± 0.013* 0.23 ± 0.01
(n＝9)
Hexadecenoic acid C16:1 0.01 ± 0.0006 0.02 ± 0.001 0.02 ± 0.002 0.03 ± 0.009*
(n＝7)
Heptadecanoic acid C17:0 0.009 ± 0.002 0.02 ± 0.003 0.03 ± 0.012 0.01 ± 0.001
Octadecanoid acid C18:0 0.09 ± 0.01 0.18 ± 0.02** 0.12 ± 0.004 0.16 ± 0.006**
Oleic acid C18:1 0.10 ± 0.01 0.34 ± 0.04**, 0.34 ± 0.01**, 0.28 ± 0.03**
Octadecenoic acid C18:1 0.06 ± 0.01 0.13 ± 0.02**, 0.17 ± 0.008** 0.2 ± 0.003
18 carbon diene C18:2,0.007 ± 0.0006 0.02 ± 0.004 0.06 ± 0.01**, 0.04 ± 0.009*
Acid
18 carbon triolefin C18:3,0.009 ± 0.001 0.01 ± 0.002 0.03 ± 0.0009 0.04 ± 0.01**
Acid
Arachic acid C20:0 0.01 ± 0.0009 0.04 ± 0.005**, 0.04 ± 0.001** 0.009 ± 0.0004
Eicosatetraenoic C20:4 0.01 ± 0.002 0.03 ± 0.007** 0.02 ± 0.007 0.03 ± 0.0008
Acid
Eicosapentaenoic C20:5 0.009 ± 0.002 0.02 ± 0.003 0.01 ± 0.001 0.04 ± 0.004**
Acid
22 carbon, four C22:4 0.01 ± 0.002 0.05 ± 0.008**, 0.03 ± 0.006* 0.02 ± 0.0007
Olefin(e) acid
22 carbon, six C22:6 0.02 ± 0.003 0.08 ± 0.02 0.11 ± 0.03*, 0.12 ± 0.03*
Olefin(e) acid
Table 10
The various free fatties that hormone causes in the rat soleus muscle of the food of the high Adeps Sus domestica content of feeding discharge
The release gas chromatograph analysis of the various free fatties that surpass 1h that caused by hormone in the food rat soleus muscle of high Adeps Sus domestica content of feeding is by the definite evaluation to various fatty acids of mass spectral analysis.Numerical value is nmol/g wwt, and is expressed as average ± S.E.M..(each n=16).* with the marked difference of comparing (P＜0.05).* and the marked difference (P＜0.01) of comparing.
Free fatty contrast amylopectin CGRP norepinephrine
Tetradecanoic acid C14:0 0.004 ± 0.05 ± 0.01** 0.03 ± 0.007* 0.04 ± 0.007**
0.0007
Pentadecanoic acid C15:0 0.04 ± 0.01 0.12 ± 0.03*, 0.14 ± 0.01**, 0.13 ± 0.02*
Hexadecanoic acid C16:0 0.1 ± 0.009 0.24 ± 0.01**, 0.24 ± 0.01**, 0.23 ± 0.02**
Hexadecenoic acid C16:1 0.02 ± 0.001 0.02 ± 0.003 0.03 ± 0.003* 0.01 ± 0.001
(n＝9)
Hexadecenoic acid C16:1 0.03 ± 0.005 0.02 ± 0.0009 0.02 ± 0.005 0.02 ± 0.0008
(n＝7)
Heptadecanoic acid C17:0 0.03 ± 0.005 0.08 ± 0.01**, 0.09 ± 0.01**, 0.08 ± 0.008**
Octadecanoid acid C18:0 0.2 ± 0.03 0.35 ± 0.04*, 0.35 ± 0.06* 0.22 ± 0.01
Oleic acid C18:1 0.17 ± 0.02 0.2 ± 0.01 0.25 ± 0.02* 0.21 ± 0.01
Octadecenoic acid C18:1 0.01 ± 0.002 0.02 ± 0.002 0.03 ± 0.006*, 0.07 ± 0.003**
18 carbon diene C18:2,0.12 ± 0.009 0.29 ± 0.004**, 0.23 ± 0.005* 0.09 ± 0.003
Acid
Arachic acid C20:0 0.02 ± 0.001 0.06 ± 0.007**, 0.07 ± 0.004** 0.03 ± 0.004
Eicosatetraenoic C20:4 0.01 ± 0.003 0.01 ± 0.0005 0.02 ± 0.001 0.012 ± 0.003
Acid
Eicosapentaenoic C20:5 0.01 ± 0.002 0.02 ± 0.002 0.02 ± 0.002 0.02 ± 0.004
Acid
22 carbon, four C22:4 0.02 ± 0.003 0.03 ± 0.005*, 0.04 ± 0.005* 0.02 ± 0.003
Olefin(e) acid
22 carbon, six C22:6 0.01 ± 0.002 0.02 ± 0.002 0.015 ± 0.002 0.01 ± 0.001
Olefin(e) acid
Amylopectin-(8-37) and CGRP-(8-37) are at the skeletal muscle of normally feeding and high lipid is fed rat
In to the influence of the hormone-mediated adjusting of lipid metabolism
A. amylopectin-(8-37) and CGRP-(8-37) in the skeletal muscle of the rat of normally feeding to the influence of the hormone-mediated adjusting of lipid metabolism
The in vitro study based on the enzymatic analysis in the stripped musculus soleus bar proves, compare with independent the cultivation with the 100nM amylopectin, 10 μ M amylopectins-(8-37) or 10 μ MCGRP-(8-37) and the collaborative intramuscular free fatty acid content that greatly reduces when using of 100nM amylopectin, be respectively that (18.5 ± 6nmol/g is to 216 ± 26, P＜0.01) and (65 ± 8nmol/g is to 216 ± 26, P＜0.01), sees accompanying drawing 3A.The peptide with antagonistic activity of these two kinds of linearities all lacks preceding 7 aminoacid, and consistent NH
2Terminal cys2-cys7 disulfide bond circulus, this circulus are that the full-length peptide of these peptides of acquisition is common.The biological agents that these two kinds of peptides all reverse and caused by CGRP and amylopectin in a large number, and with different effectiveness in various tissue with corresponding radiolabeled analog competition.(Wang etc.1991, FEBS Lett291:195-198; Chantry etc., 1991, journal of biological chemistry 277:139-143; Beaumont etc., 1998, U.S. physiology magazine 274:F51-62; Aiyar etc. 1995, neuro chemistry magazine 65:1131-1138; Bhogal etc. 1994, peptide 15:1383-1390; Perry etc., 1997, endocrinology 138:3486-3496; Zimmermann etc., 1997, endocrinology magazine 155:423-431).With respect to only cultivating with 100nM CGRP, amylopectin (8-37) suppresses CGRP-100nM and increases the ability of the free fatty acid content in the musculus soleus (34 ± 5nmol/g than 334 ± 52, P＜0.01), sees accompanying drawing 3B.CGRP antagonist CGRP-(8-37) also suppresses the rising (90 ± 7nmol/g to 334 ± 52, P＜0.01) of the free fatty that caused by 100nM CGRP.The adding of the insulin of maximal stimulus has suppressed the ability that 100nM amylopectin (216 ± 26nmol/g than 100 ± 52, P＜0.01) or CGRP (334 ± 52nmol/g than 107 ± 17, P＜0.01) promote the increase of free content of fatty acid in the musculus soleus.Therefore, these two kinds of functionss of hormones of insulin resistance cause the release of free fatty acid in the skeletal muscle.
When CGRP concentration is 1pM, 100pM CGRP-(8-37) and amylopectin-(8-37) can significantly reduce the free-fat acid concentration in the musculus soleus, are respectively (68 ± 8nmol/g than 166 ± 18, P＜0.01) and (86 ± 20nmol/g is than 166 ± 18, P＜0.01), sees accompanying drawing 3C.
This research provides positive evidence,
8,37Amylopectin and
8,37CGRP can suppress the intramuscular free fatty that caused by amylopectin and CGRP and raise, promptly as the competitive antagonist of composing type CGRP/ amylopectin receptor.By the deutero-antagonist of amylopectin
8,37The K of the composing type receptor of amylopectin
iBe significantly higher than the deutero-antagonist of corresponding C GRP
8,37CGRP.The difference of about 20 times binding affinity, this quantitatively with
8,37It is consistent to doing of raising of glucose that CGRP more effectively blocks amylopectin.Confirmed 1 μ M and 10nM
8,37The inhibition that CGRP counter-rotating is raise by the glucose to insulin stimulating of 10nM amylopectin mediation, with observed these peptides to composing type C1
Ins-The binding affinity unanimity of/ramp 1 receptor.Therefore, the antagonist of mensuration
8,37CGRP and
8,37Amylopectin is to the C1 in the L6 sarcoplast
Ins-The binding affinity of/ramp 1 receptor is consistent with its relative potency in the musculus soleus that exsomatizes.Similarly,
8,37The cross-reactivity of CGRP and this antagonist amylopectin that reverses competitively suppresses and amylopectin causes the ability consistent (Wang etc., 1991, FEBS Lett 291:195-198) of blood-glucose and lactic acid rising the insulin stimulating glycogen is synthetic.The order of the effect of antagonist be antagonist order with CGRP and amylopectin by with inherent C1
Ins-/ ramp 1 receptors bind and cause hypothesis unanimity to the effect of skeletal muscle glucose metabolism.
The influence of the rat muscle lipid that B.CGRP receptor antagonist counter-rotating CGRP-1 feeds to high lipid
The Wistar rat is from weaning period (21 days) the high lipid food (40% Adeps Sus domestica) 30 days (up to 51 days) that begins to feed, when lacking or existing CGRP antagonist, amylopectin-(8-37) or CGRP-(8-37), the rat soleus muscle 1h that exsomatizes with the CGRP1 In vitro culture.Extract and the quantification TL.The result is expressed as the NEFA of accompanying drawing 18A and the triglyceride of 18B.
In the rat of normally feeding, the effect of CGRP receptor antagonist counter-rotating CGRP.The reduction of the content of triglyceride that is caused by 1pM or 100nM CGRP1 is suppressed by any antagonist of 10 times of molar excess.For the high-affinity site, the 100pM antagonist is enough to improve the influence of 1pM CGRP to triglyceride in the musculus soleus and free fatty acid content.
Amylopectin is to stimulating the effect of the dosage-dependence of free content of fatty acid in the rat soleus muscle of cultivating
CGRP is to stimulating the effect of the dosage-dependence of free content of fatty acid in the rat soleus muscle of cultivatingThe data of CGRP-1 dose response research are shown in accompanying drawing 4A and 4B.These studies show that and have two kinds of concentration ranges, and wherein the free fatty acid content in the CGRP rat soleus muscle that causes cultivating reduces.The EC of phase I
50Be worth is 0.25pM ± (fiducial range of 13-0.5pM[95%], corresponding r
2Value is 0.84.The EC of second stage
50Be worth is 45nM ± (8.5-95), and corresponding r
2Value is 0.51.Data from CGRP-2 research are shown in accompanying drawing 4C.This studies have shown that CGRP-2 has the mechanism of action that is different from its analog, and is in fact similar to amylopectin.EC
50Value is 5.2nM ± (2.3-11.5; Average ± 95%C.I.), corresponding r
2Value is 0.78.
Amylopectin is to stimulating the effect of the dosage-dependence of free content of fatty acid in the rat soleus muscle of cultivatingAmylopectin acts among the accompanying drawing 5A the dosage-dependence that stimulates the content of fatty acid that dissociates in the rat soleus muscle of cultivating and illustrates.The progressively increase of amylopectin concentration has improved the content of the free fatty in the rat soleus muscle in the culture fluid.The EC of this effect
50Be 7.8nM ± (4.1-14.7, average ± 95%C.I.), corresponding r
2Value is 0.87.
When having CGRP (100pM), free fatty acid contains amylopectin in the rat soleus muscle of cultivating to stimulating
The effect of the dosage-dependence of amount
The amylopectin dose-effect curve that accompanying drawing 5B obtains when representing to have 100pM CGRP.EC
50Be worth is 11.6nM ± (4.8-27, average ± 95%C.I.), corresponding r
2Value is 0.79.
Free fatty from skeletal muscle after the HORMONE TREATMENT calculates 11 expressions of metabolizable energy scale from using side chainStarch, CGRP and norepinephrine are handled the metabolisable energy of the free fatty acquisition that discharges behind the musculus soleus 60min.Aggregate level derives from the GC data.The quantity of the potential energy of various free fatties is calculated (Mathews etc., 2000, biochemistry, the 3rd edition, BenjaminCummings Pub.Co., California) according to the possible ATP output of mitochondrion fatty acid beta-oxidation fully.The Conversion of energy of these calculation assumptions ideal 100%.Amylopectin and CGRP cause that the intramuscular free fatty acid content increases, and can obtain more potential energy (2.73 ± 0.28 μ mol/g to 1.97 ± 0.31, P＜0.01) with respect to contrast, (2.96 ± 0.22 μ mol/g to 1.97 ± 0.31, P＜0.01).After handling, there is not marked difference between potential metabolisable energy that obtains from free fatty and the contrast (2.24 ± 0.17) with norepinephrine.
Table 11
The metabolism potential that free fatty after HORMONE TREATMENT obtains
By total release of the free fatty that causes by hormone in the gas Chromatographic Determination musculus soleus, determine evaluation to various free fatties by mass spectral analysis.Various free fatties are used to calculate by free fatty and transform the theoretical metabolisable energy that obtains by the beta-oxidation of mitochondrion fatty acid fully.The energy conversion efficiency of these value of calculation supposition 100%.Numerical value is average ± S.E.M (each n=16).All hormone concentrations are 100nM.* is marked difference (p＜0.01) compared with the control.
Contrast amylopectin CGRP norepinephrine
Total free fatty 0.467 ± 0.03 0.636 ± 0.05**, 0.691 ± 0.04** 0.527 ± 0.03
(nmol/g?wwt)
Obtain energy (μ 63.56 ± 5.2 88.2 ± 9.08 95.55 ± 7.28 72.58 ± 5.58
mol?ATP/g/hr)
(kJ/g/hr)??????1.97±0.31?????2.73±0.28**?????2.96±0.22**?????2.24±0.17
Materials and methods
Present embodiment is described in materials and methods all in the test of describing among the embodiment 8-14, comprises the cell of preparation express recombinant receptor.
Peptide, radiochemicals and other analytical reagentThe sequence of the sequence of AC128 and Muridae amylopectin (Cooper etc., 1994, endocrinology research 15:163-201,1994) is identical, is called " amylopectin " (catalog number (Cat.No.) 0538912) hereinafter; Rat CGRP-1 (' CGRP '; Catalog number (Cat.No.) 501460); Rat calcitonin (catalog number (Cat.No.) ZJ375); Salmon calcitonin see calcimar (' sCT '; Catalog number (Cat.No.) ZM423);
8,37Rat CGRP-1 (catalog number (Cat.No.) 501395) is available from Bachem (Switzerland).People adrenomedullin is according to previous description synthetic (Heller etc., 2000, analytical chemistry 285:100-104).Determine that by mass spectral analysis (ground substance assistant laser absorbs ionization time flight mass spectrum, MALDI-TOF for all peptide prepared product integrity and purity; Hewlett-Packard G2025A; 4-hydroxyl-styrene acidic group matter).Soluble insulin human all is used as igf agonist (' insulin '; Actrapid 100U, Novo Nordisk).Synthetic peptide is stored in the argon up to use under-80 ℃ with powdered in batches.Be dissolved in the dual deionized water of 18M Ω, obtain mother solution, as required, mother solution is added the specified ultimate density of acquisition in the reactant liquor.[2-
3H] 2-deoxy-D-glucose ([
3H] 2-DOG; Catalog number (Cat.No.) 2711-158,30.6Ci/mmol) and D[
14C (U)] (catalog number (Cat.No.) 2643-317 is 10.6mCi/mmol) available from New England Nuclear for glucose; D[
14C (U)] Sorbitol available from U.S. radio-labeled chemical drugs company (American Radiolabeled Chemicals Inc) (catalog number (Cat.No.) 950314,320mCi/mmol); [α-
32P] dCTP is available from Amersham (catalog number (Cat.No.) A025694; 3000mCi/mmol).Unless otherwise noted, every other reagent is AG.
Synthetic and the analysis of radiolabeled hormone analogsCGRP, amylopectin and sCT by reductive methylation with [
3H] NaBH
4Carry out radio-labeled, and purification as discussed previously then (Cornish etc., 1997, Am.J.Phys.36:E1113-E1120).By gel filtration, follow the RP-HPLC purification
3The peptide of H labelling, with MALDITOF-MS determine [
3H] insertion of methyl labelling.Measure [
3H] given activity of radioligand is: CGRP, 26.2GBq/mmol; Amylopectin, 24.0GBq/mmol; And sCT, 23.2GBq/mmol.In argon ,-80 ℃ of storages down, radioligand can be stablized above 12 months when dryly.
Reverse infix form (the C1 of Muridae ramp 1, Muridae ramp 3 and Muridae calcitonin Ins- ) clone's branchSub-biological method is described (Sambrook J, 1989, molecular cloning: test chamber handbook, cold spring lane test chamber publishing house).Ramp 1 and Ramp3 (the genbank accession number is respectively AA544629 and AF146524) clone through RT-PCR from total RNA of Muridae musculus soleus (1 μ g) from extracting.At 65 ℃ of following heating 10min, and with hexabasic basic sequence (hexamer) initial (Primed) at random.Use Expand
TM-RT is at 30 ℃ of following reverse transcription 10min, then at 42 ℃ of following 45min.Reaction condition is: tri-HCl (50mM); KCl (40mM); MgCl
2(5mM); Tween-20 (0.5%); Dithiothreitol, DTT (10mM); RNA enzyme inhibitor (20U); Expand
TMReverse transcription (50U) (Boehringer Mannheim); DNTP ' s (1mM); PH 8.3.The oligonucleotide primers of Ramp1 and Ramp3 is respectively
5-TTA
GGATCCGTTGCCATGGCCCGGCTGCGGCTCCCG-3 (forward)
/ 5-CGA
GAATTCCTCATCACCCGGATACCTA-3 (oppositely)
And 5-ATA
GGATCCTGCATCTTAGTTGGCCATGA-3 (forward)
/ 5-ATA
GAATTCATCCAGCAGATCCTCAAGC-3 (oppositely).The nucleotide of underscore represents to be introduced into convenient clone's BamH I and the restriction site of EcoR I respectively.Pcr amplification carries out at Eppendorf MastercyclerR gradient thermal cycler, 94 ℃ (1min), 55 ℃ (2min) and 72 ℃ (3min) circulation 40 times.Reaction condition is: Tri/HCl (10mM); KCl (50mM); MgCl
2(1.1mM); Oligonucleotide (0.5 μ M), gel (0.01%w/v), dNTP ' s (200 μ M), U REDTaq
TMArchaeal dna polymerase (Sigma, Saint Louis), cDNA template (10ng), pH 8.3.Separate the PCR product, the restriction site sub-clone by BamH I and EcoR I is in pcDNA3.1+ (Invitrogen).Sequence analysis confirms the fidelity (fidelity) of Ramp 1 and Ramp3 sequence.
From the total RNA of Mus brain, clone the calcitonin receptor C1 of reverse infix form by RT-PCR
Ins-(Genbank accession number U18542), used oligonucleotide primers are 5-CCATCCCTGCCTGCAGATGC-3 (forward) and 5-CTCTATTTCTAGACTGACTCCC-3 (oppositely); The base of underscore is introduced into convenient clone's BamH I and the restriction site of EcoR I, and the black matrix base represents to introduce the point mutation of restriction site.Use Elongase
TMCarry out RT-PCR.94 ℃ (30s), 56 ℃ (30s), 68 ℃ (2min) circulation 5 times, circulate down at 94 ℃ (30s), 60 ℃ (30s) and 68 ℃ (2min) then and increase for 30 times.Will be corresponding to calcitonin receptor C1
Ins-The PCR product sub-clone of 1.9kbp to pGEM-T (Promega), and then with EcoR I and Xba I enzyme action.Will be from C1 by EcoR I and Xba I site through dual clone's step
Ins-Two fragments that obtain by sub-clone to pCDNA3.1.Muridae C1
Ins-The fidelity of sequence is determined by sequence analysis.Also the sequence of clone's product is carried out RT-PCR and clone and show, corresponding to C1
Ins-RNA in rat soleus muscle, express, this RNA is the main RNA kind of corresponding detected calcium in the musculus soleus.(result does not show).
The bonded detection of the transfection of L6 sarcoplast and cos-7 cell and radioligandL6 sarcoplast and cos-7 cell derive from ATCC.At 5%CO
2Under/95% air and 37 ℃, two kinds of cell lines are at the improved Eagle culture fluid of Dulbecco (DMEM; Gibco/BRL, high glucose) the middle cultivation, added 10% hyclone (Gibco/BRL in the culture fluid, Gaithersburg, MD), L-glutaminate (2mM), penicillin (100U/ml, Sigma), streptomycin (100U/ml, Sigma), normal saline (0.85%).Transfection the previous day, the cell trypsinization is then with 2.9 * 10
5Cells/well is implanted in the identical culture fluid of six well culture plates.During transfection, cell with contain the various Qiagen purification of 1 μ g (Qiafen, Valencia, CA) contain C1
Ins-Plasmid and Fugene reagent (6 μ l with ramp 1 or ramp 3; Boehringer Mannheim) Opti-Mem I solution (Gibco/BRL) mixture is cultivated together.Complex adds in the cell with serum, cultivates 48h again.
For in conjunction with test, radioligand before the test (individually or with the nonradioactive labeling's who specifies ultimate density part) is dissolved in (binding buffer liquid) 1h among the DMEM that contains 0.1%BSA.Select according to previous time-histories test (data not shown) in conjunction with condition.Cell is cultivated 4h with the same buffer that adds radioligand down in room temperature (21 ℃) then with sort buffer liquid washed twice.Then, cell carry out twice washing transfer with 0.5M sodium hydroxide pair cell after, is counted the radioactivity of combined cell with ice-cold binding buffer liquid washed twice by the liquid scintillation spectrophotometer.
The Northern blot hybridization is analyzedUse Trizol
TMReagent (Life Technologies) according to manufacturer's recommended hour from total RNA.Upward total RNA (5 μ g/ swimming lane) is carried out electrophoresis at formaldehyde/agarose gel (1%w/v), transfer to Nylon Hybond
TMOn the film (Amersham Pharmacia Biotech), then with Stratalinker 2400 (Stratagene, La Jolla, CA) crosslinked.Mice ramp 1 and ramp 3 cDNA hybridization probes usefulness [α-
32P] dCTP is initial at random.Filter is 65 ℃ of following prehybridization 2h in Church-Gilbert solution (0.25M sodium hydrogen phosphate, 7%SDS, 1mM EDTA), hybridize under 65 ℃ in the same buffer of the probe that contains the 25ng/ml labelling then and spend the night.Filter washs 30min twice under 65 ℃, in 2 * SSC, then wash 30min twice in 1 * SSC, 0.1%SDS and 1 * SSC, 0.5%SDS.The Northern trace is visual by the phosphor pattern instrument.
The preparation of musculus soleusAll tests are carried out according to the method for public animal Ethics Committee (Institutional AnimalEthics Committee) approval.The male Wistar rat (160g) in age in 5-6 week is in 12: 12 illumination/dark cycle, free choice feeding and drinking-water.Rat is put to death by taking off neck then with 50mg/kg Nai Bota (pentobarbital sodium) anesthesia.At oxidation KHB (118mM NaCl, 4.75mM KCl, 1.2mM MgSO
4, 24.8mM NaHCO
3, 1.2mM KH
2PO
4, 2.54mM CaCl
2, the 10mM glucose, dissect musculus soleus in pH7.4), and be torn into half-and-half following then cultivation.
Determine the cAMP in cultured cell and the musculus soleus barMeasure the cAMP concentration in the also transfected cell of implanted as described six well culture plates.After feeding gas, cell is cultivated 15min under the room temperature in having the binding buffer liquid of prescribed concentration part.Add isobutyl methylxanthine (Sigma) to the ultimate density of 0.5mM and the peptide of prescribed concentration, feed gas then and finish reaction.(10mMTris/HCl, 1mM EDTA pH7.6) wash, and transfer to rapidly in the 1ml same buffer of centrifuge tube cell, heating 5min with ice-cold TE buffer.With 12, behind the centrifugal 5min of 000g, in cooled on ice, identical 50 μ l aliquots adopt based on competition bonded analytical system (Amersham Pharmacia), carry out cAMP according to manufacturer's recommendation and analyze under 4 ℃.
For the cAMP that measures in the muscle, the musculus soleus bar that exsomatizes is cultivated with the peptide of prescribed concentration, then lyophilizing and deproteinization.Each strip of muscle is placed on the HClO that 400 μ l contain 5mM EDTA
4In (phosphodiesterase inhibitor) and handle 3 times with the pulse ultrasonic wave of 20s.Test tube heats 3-5min and comes coagulated protein in boiling water, centrifugal then (12,000g,, 4 ℃, 2min).Get supernatant (300 μ l), with 75 μ l 2M NH
4It is 7.0 that OH regulates pH.It is freezing that (4 ℃, 2h) sample comes protein precipitation, and is centrifugal under condition same as described above then, and gets 50 μ l and be used for cAMP and measure.
By the mensuration of glucose transport speed in the musculus soleus bar of cultivatingContain at each and to add 4-5 strip of muscle in the culture bottle of 10ml DMEM.With specified hormone 30 ℃ of pre-down 30min that cultivate in rocking water-bath, after this, add 5 μ l 2[
3H] DOG (0.5 μ Ci/ml) and 5 μ l D[
14C (U)] Sorbitol (0.5 μ Ci/ml), and then cultivate 30min; The BSA to 0.9% (w/v) that adds the RIA level stops that insulin is non-to be attached on the culture bottle specifically.After the cultivation, with strip of muscle transfer to 5-10min among the ice-cold KHB remove excessive [
3H] DOG and [
14C] Sorbitol.Then trace, freezing, the excision tendon, lyophilizing 48h again.The muscle of weighing is then transferred in the scintillation vial, with 0.5ml 1M NaOH at 60 ℃ of digestion 1h down.Add scintillation solution (4ml) in each pipe, sample is set to count simultaneously at Beckman liquid scintillation spectrophotometric random encounter number
14C and
3The H signal.Determine to be present in the various isotopic amount in the sample, be used to calculate the extracellular and intracellular [
3H] DOG concentration.The measured value that the muscle glucose raises be in the cell [
3H] accumulation of DOG, by from corresponding total muscle [
3H] deduct among the DOG extracellular [
3H] concentration of DOG calculates; Extracellular [
3H] DOG by measure in the muscle [
14C] concentration of Sorbitol quantizes.
Measure the glycogen metabolism in the stripped musculus soleus of cultivatingFor D[
14C (U)] conversion of glucose is the mensuration of muscle glycogen, strip of muscle is the pre-30min that cultivates in DMEM, then when lacking or having the 23.7nM insulin, with 0.5 μ Ci D[
14C (U)] glucose and amylopectin, CGRP and sCT cultivate 2h with prescribed concentration.Freezing rapidly, lyophilizing, the strip of muscle of weighing, 70 ℃ of digestion 45min (the dry strip of muscle of 5mg/0.4ml 10.7M potassium hydroxide) down in 60% (w/v) potassium hydroxide then, and irregularly mix.Glycogen precipitates down at-20 ℃ with 96% ethanol (1.2ml) and spends the night, then with 8, and 000g centrifugal (15min, 4 ℃).With equivalent washing with alcohol precipitate twice, be dissolved in then (0.6ml) in the water.Aliquot (0.3ml) is analyzed D[by the liquid scintillation spectrophotometer
14C (U)] glucose content.
By the lyophilization strip of muscle, then measure total glycogen content for the D-glucose with amyloglucosidase (Sigma) the hydrolysis glycogen of the 20U/100ml among 0.2M sodium acetate/acetate buffer pH7.4, be expressed as μ mol ' glucityl ' unit/g muscle dry weight.With the free glucose of the fixed enzymology medicine of D-glucoseoxidase in mensuration supernatant in glucose/lactic acid analyser (YSI 2300 STAT, Yellow Springs Corporation).
Data analysisAdopt the significance,statistical of single factor ANOVA, the check of Dunnett multiple comparisons and not paired t-check calculated difference aptly.Dose-response curve and the match of S shape dose response algorithm, as follows: raise for 2-DOG,
With for total glycogen and [
14C] conversion of glucose adopts four parameter logarithmic models,
(CA) row calculates and obtains the maximum valid density (EC of half for Graph Pad software, San Diego with Prism v2.0
50).Calculate the displacement curve of in transfectional cell, not competing with Prism v2.0 according to a site competitive way with radiolabeled part and bonded radioligand.
Be attached to Muridae ramD 1 and C1
Ins-
The myoblastic radioligand of the L6 of cotransfection
Be in the news, when the calcitonin receptor of reverse infix form and people ramp 1 or ramp 3 isotypes (isoform) coexpression, receptor response (Muff etc., 1999, endocrinology 140:2924-2927 to amylopectin take place; Christopoulos etc., 1999, molecule pharmacy (Mol.Pharmacol) 56:235-242).This paper, the inventor measure the character of corresponding Muridae receptor isotype, as Muridae ramp 1 and Muridae C1
Ins-(accompanying drawing 8) forms this isotype during cotransfection L6 (Muridae) sarcoplast.Separately with carrier (accompanying drawing 8A), ramp 1 (accompanying drawing 8B) or C1
Ins-Do not detect the specific bond of significant amylopectin in (accompanying drawing 8C) cells transfected, and in separately with ramp 1 cells transfected, sCT shows lower still significant specific bond (accompanying drawing 8C), on the contrary, when using ramp 1 and C1
Ins-During the construction cotransfection, the remarkable increase (accompanying drawing 8D) of the specific bond of amylopectin and sCT takes place.Time-histories test determine [
3H] amylopectin is in conjunction with reaching the required cultivation period of balance (result does not show).
Bonded from the L6 sarcoplast of transfection [
3
H] displacement of amylopectin
At C1
Ins-In the L6 sarcoplast of ramp 1 cotransfection, with CGRP-1 and amylopectin displacement specific bond [
3H] amylopectin, have similar K
iValue and EC
50Value (accompanying drawing 9A).This finds to compete the lip-deep individual molecule of pair cell sites in conjunction with consistent with two kinds of peptides.On the contrary, sCT has higher affinity and lower K
I, higher in conjunction with the consistent (table 12 of ratio with it; Accompanying drawing 8C and D).Rat calcitonin and people adreomedullin, the 5th member's representative in this peptide family only very faintly competed the cell (accompanying drawing 9A) that combines cotransfection with amylopectin.Different with the similar bonded characteristic of full-length peptide, as the flush end peptide of the linearity of antagonist
8,37Amylopectin and
8,37Marked difference (the table 12 that occurs binding affinity between the CGRP; Accompanying drawing 9B).
8,37CGRP and the bonded K of these cells
iRatio
8,37Amylopectin hangs down about 20 times (accompanying drawing 9B).The IC that measures
50With the K that obtains
iValue is summarised in the table 12.The K of the AC128 that this test obtains
iValue (table 12) with from saturation testing, obtain [
3H] K of amylopectin
dValue (9.2 ± 0.2nM, average ± S.E.M; B
Max=1.5
Table 12
In the L6 of cotransfection sarcoplast, by calcitonin family peptides/antagonist and Muridae ramp 1/C1
Ins-
Receptor obtains
Incorporating parametric
R
2???????????IC
50(nM)???????????K
i(nM)
Salmon 0.96 1.9 (0.9-2.5) 0.5 (0.3-8)
Calcitonin CGRP 0.96 63 (43-93) 20 (13-29)
Amylopectin 0.92 23 (13-44) 7.4 (4-14)
8，37CGRP???????????????0.92??????????62(35-108)??????????19.5(11-34)
8,37Amylopectin 0.87 1270 (661-399 (208-763)
2420)
With the radioligand concentration of 22nM and the K that obtains from saturation curve independently
dValue 9.2 is by the method calculating K of Cheng and Prusof (Cheng etc., 1973, biochemical pharmacology (Biochem.Pharmacol.) 22:3099-3108)
iValue.Comprise 95% confidence interval (nM).
The influence of the cAMP output that peptide antagonists causes the CGRP in the transfected L6 sarcoplast and amylopectin
In order to inquire into effect, for the ramp 1/C1 that forms as the antagonist of the signal conduction that causes by peptide
Ins-The receptor phenotype, research
8,37CGRP and
8,37The influence (accompanying drawing 10) that amylopectin generates the cAMP that is caused by CGRP and amylopectin.CGRP and amylopectin cause all that cAMP generates in the sarcoplast of cotransfection dosage relies on to be increased, though tested under the concentration at all, the size of this reaction is significantly less than amylopectin.
Tested at all that (10pM is to 100nM under the concentration; Accompanying drawing 10A), 5 μ M
8,37CGRP eliminates the cAMP that is caused by CGRP and raises.This concentration is enough to saturated receptor, than the CGRP that measures and the K of amylopectin
iBe worth big approximately 250 times.With CGRP and
8,37K between the CGRP
iThe similarity unanimity of value, used maximum concentration CGRP (100nM) is excessive by 50 times
8,37CGRP (5 μ M) competes effectively.On the contrary, 5 μ M
8,37Amylopectin is significantly lower as the efficient of amylopectin antagonist, only causes that the appropriateness of cAMP output reduces.
Using ramp 3 and C1
Ins-
Radioligand after the transfection in conjunction with cell
With Muridae ramp 3 and C1
Ins-The cotransfection sarcoplast can not produce specific bond (data not shown) significant and any used radioligand.Be in the news, the cotransfection of cos-7 cell can produce [
125I]-AC128 or [
125I] human branched chain starch and C1
Ins-/ ramp 1 and C1
Ins-Combination (Muff etc., 1999, the endocrinology 140:2924-2927 of/ramp 3 receptor isotypes; Christopoulos etc., 1999, molecule pharmacy 56:235-242).Therefore, the inventor compares test with corresponding Muridae isotype in this cell.C1 of the present invention
Ins-The cotransfection of/ramp 1 and disclosed result similar (accompanying drawing 11A-C).But, consistent with result in L6, use Muridae C1
Ins-/ ramp 3 transfections can not obtain the specificity combination.By transcribe (accompanying drawing 12, the right group) of ramp 3 in the definite cos cell of Northern analysis, less with respect to transcribing of ramp1 (accompanying drawing 12, left side group) ramp 3.But, in this system, in the finite concentration scope, do not observe the variation (data not shown) of the efficient of transcribing by ramp 3 plasmids.But the cos-7 cell of untransfected have less significantly to all three kinds [
3H] the intrinsic specific bond of peptide, show that they express low-level public receptor.In fact, RT-PCR shows that L6 and cos-7 cell all express C1
Ins-(data not shown), the difference that viewed binding affinity is described may be because ramp1 and the ramp 3 inherent changes of expressing.
CGRP and amylopectin are to the influence of cAMP content in the musculus soleus
Relation in the skeletal muscle between CGRP/ amylopectin signal and the cAMP metabolism is quite disputed on, and part is because inconsistent between the different demonstration tests.The early stage amylopectin that studies show that can not influence the content of cAMP in the skeletal muscle, with existence and irrelevant signaling mechanism consistent (Deem etc., 1991, the biochemistry biophysical studies communication 174:716-720 of cAMP; Kreutter etc., 1993, U.S. physiology magazine 264:E606-613).These find with other opposite to cAMP in the skeletal muscle and the active potential stimulus report of adenylyl cyclase (Pittner etc., 1995, Biochimical Biophys.Acta1267:75-82) about amylopectin.
A) CGRP of nanomole concentration and amylopectin cause that by metabolism amylopectin receptor cAMP generates
In this test, measure CGRP (accompanying drawing 13A) and amylopectin (accompanying drawing 13B) interaction in vitro to cAMP generation in the musculus soleus, and with they to using C1
Ins-The influence (accompanying drawing 10A and B) that cAMP generates in the cell of/ramp 1 cotransfection relatively.Show that amylopectin reaches maximum to influence 5min after using amylopectin of cAMP content in the musculus soleus and (increases by 2 times, p＜0.01, data do not provide), this time-histories to the reaction of isopropyl noradrenalin (beta-adrenaline excitant) in the perfusion isolated heart with previous report has comparability.When lacking and have insulin (23.7nM), the CGRP of 5nM and 10nM causes the remarkable rising (accompanying drawing 13A) of muscle cAMP concentration.When using with 10nM and 100nM, amylopectin causes that the maximum of cAMP content increases about 2 times (accompanying drawing 13B); The size of this effect (23.7nM) when lacking and have insulin does not have difference.The amylopectin of picomole concentration has illustrated active difference between amylopectin and the CGRP-1 (accompanying drawing 13C) to the not influence of muscle cAMP level.
This biological action of CGRP and amylopectin and they stimulate by C1
Ins-The effect unanimity of the cAMP content in the L6 sarcoplast of/ramp 1 cotransfection (accompanying drawing 10).And, competitive antagonist
8,37Amylopectin passes through C1 with amylopectin to the inhibition (accompanying drawing 9B and 11B) of the cAMP stimulation of amylopectin mediation in physiological muscle
Ins-The phenomenon of this effect that/ramp 1 receptor causes is consistent.
B) CGRP1 of picomole concentration causes that by high-affinity receptor cAMP generates
In the musculus soleus of the animal of normally feeding, 100nM CGRP (6.9 ± 0.8 couples of 3.9 ± 0.2pmol/mg, P＜0.01) and epinephrine (9.6 ± 0.7 couples of 3.9 ± 0.2pmol/mg, P＜0.01) cause the remarkable increase of cAMP content.CGRP of 10 times of molar excess (8-37) (4.8 ± 0.2 couples of 6.9 ± 0.8pmol/mg, P＜0.01) or amylopectin-(8-37) (2.9 ± 0.1 couples of 6.9 ± 0.8pmol/mg, P＜0.01) suppress this increase.After cultivating 1h, do not observe the remarkable increase of cAMP with 1pM CGRP.From the animal of the high lipid of feeding, separate musculus soleus, 100nM CGRP 1 (9.3 ± 1 to 5.7 ± 0.2pmol/mg, P＜0.001) also causes the remarkable increase of cAMP content, the (4.3 ± 0.8pmol/mg of the amylopectin that these can be by 10 times of molar excess-(8-37); P＜0.001) or CGRP-(8-37) (5.4 ± 0.4; P＜0.001) suppresses.But, opposite with the musculus soleus that derives from the animal of normally feeding, cultivate significantly increase cAMP content (8.6 ± 0.4 to 5.7 ± 0.2pmol/mg, P＜0.001) with 1pM CGRP.The amylopectin of 10 molar excess-(8-37) or CGRP-(8-37) are reduced to 4.4 ± 0.4pmol/mg (P＜0.001) and 4.7 ± 0.5pmol/mg (P＜0.001) with cAMP content from 8.6 ± 0.4pmol/mg respectively, see accompanying drawing 19B.
Between the animal of normal and the high lipid food of feeding, the baseline cAMP content significant difference (5.7 ± 0.2 couples of 3.9 ± 0.2pmol/mg, P＜0.01) of static musculus soleus.
Competitive antagonist is to the effect of amylopectin mediation inhibition glucose transport
After cultivating 30min, measure amylopectin to the effect baseline in the musculus soleus and glucose transport insulin-mediated, be reported in the table 13 with 2-DOG.Handle the rising that can not cause glucose significantly with amylopectin separately, and 3.2 times (P＜0.01) of insulin stimulating glucose rising.On the contrary, amylopectin (10nM) makes the glucose of insulin stimulating reduce by 31%, reaches the numerical value with the baseline difference with insignificance.
8,37Amylopectin and
8,37CGRP in various tissues, all suppress CGRP and amylopectin effect (referring to Cooper, 2001, physiology's handbook, the 7th chapter: hormonal system.II volume endocrine pancreas and metabolic regulation (AD edits for Jefferson, LS Cherrington), the Oxford University Press).In this article, the musculus soleus bar adds at external use amylopectin (10nM) and insulin (23.7nM) and specifies ultimate density
8,37Amylopectin and
8,37CGRP cultivates (accompanying drawing 14) in advance.1 μ M and 10 μ M's
8,37CGRP antagonism amylopectin effectively is elevated to 30.4 ± 3.0 (n=4, p＜0.05) to insulin stimulating ground glucose rising ground inhibitory action (accompanying drawing 14A) by 19.5 ± 3.6nmpl/g dry weight muscle/min (n=5).On the contrary, even be 10 μ M,
8,37Amylopectin is not to significantly acting on (accompanying drawing 14B) by amylopectin mediation glucose is raise.
Table 13
In the stripped musculus soleus cutlet of cultivating with hormone, AC128 is to baseline (no insulin) or maximum
The glucose rising ratio that amount (23.7nM) stimulates is measured by 2-deoxy-D-glucose.
2-DOG raises
(the significance level of nmol/g muscle dry weight/min)
Baseline 8.8 ± 0.9 (n=3)-
Amylopectin (10nM) 13.4 ± 2.4 (n=5) p＞0.05
Insulin (23.7nM) 28.4 ± 6.4 (n=3) * * P＜0.001
Amylopectin (10nM)+insulin 19.5 ± 3.6 (n=5) p＞0.05
(23.7nM)
Carry out statistical analysis with single factor ANOVA, then analyze with Dunnett multiple comparisons check carrying out post hoc.Significance level is meant the comparison between the ratio of the ratio of hormone-treated muscle and base line condition.The quantity of n=independent trials.
CGRP and amylopectin are to the comparison of the effect of glycogen metabolism in the musculus soleus
The expection test shows in stripped flatfish muscle former generation of total sugar and D[when insulin concentration is 23.7nM
14C (U)] conversion of glucose is glycogen the highest (data not shown).Therefore, lack or when having this concentration insulin, after cultivating 2h, measure and improve the effect (accompanying drawing 15) of CGRP, amylopectin and sCT concentration glycogen content.Concentration-the response curve of glycogen content shows that the 10nM amylopectin causes a large amount of consumption (accompanying drawing 15A) of total glycogen content, from baseline ratio to 65.6 ± 9.1 (n=4 of 90.8 ± 3.7 μ mol/g muscle dry weight/h (n=15); P＜0.05).10nM CGRP also sees the similar reduction of glycogen content, is 71.4 ± 7.7 μ mol/g muscle dry weight/h, although this difference also fail to reach statistical significance (n=4, p=N.S.).When having insulin, the inhibitory action of these two kinds of peptides all more remarkable (accompanying drawing 15B).Handle with the 10nM amylopectin, total glycogen content is reduced to 86.7 μ mol/g muscle dry weight/h (n=4, p＜0.01) from 138 ± 4.8 μ mol/g muscle dry weight/h (n=12).On the contrary, only make glycogen content be reduced to 113 ± 6.9 μ mol/g muscle dry weight/h (p=N.S.) with 10nM CGRP cultivation.Therefore, amylopectin to the maximum effect of muscle glycogen content significantly greater than the effect of CGRP.SCT does not have significant difference (accompanying drawing 15A and B) to the effect of glycogen content and the effect of CGRP.
D[
14C (U)] conversion of glucose is that the ratio of glycogen is the synthetic measured value of external glycogen in the skeletal muscle.Therefore, with D[
14C (U)] glucose and peptide be added in the culture fluid together, cultivate 2h after, in strip of muscle, measure the ratio that is converted into glycogen.When lacking amylopectin, handle the conversion ratio that reduces glucose with the 10nM amylopectin, from 4.8 ± 0.3 μ mol/g muscle dry weight/h (n=16) to 2.7 ± 0.8 (n=5, p＜0.01; Accompanying drawing 15C).Similarly, 10nMCGRP makes conversion of glucose be reduced to 2.8 ± 0.3 (n=7, p＜0.01).When having the Cmax insulin, the 10nM amylopectin reduces the conversion (accompanying drawing 15D) of radiolabeled glucose, from 27.6 ± 1.4 (n=11) to 16.0 ± 2.5 (n=4, p＜0.05), wherein same concentrations CGRP suppresses conversion of glucose, be 13.7 ± 0.7 μ mol/g muscle dry weight/h (n=, p＜0.01).When lacking insulin, to the reaction comparison CGRP of sCT or to the reaction of amylopectin more effectively (accompanying drawing 15C) significantly, when wherein having insulin, the reaction of sCT and the reacting phase of CGRP same (accompanying drawing 15D).When the muscle glycogen content descends, take place by all three kinds of consumption that peptide causes to measure in conversion of glucose, thereby the synthetic ratio of expression glycogen reduces.
From these concentration-response curves, draw EC
50Value and 95%CI (confidence interval) (table 14).When having insulin, the amylopectin effect significantly is lower than other two kinds of peptides.Observe between the peptide about the synthetic maximum marked difference that suppresses of glycogen.These results show there is to a certain degree inhomogeneities that between receptor subtype these three kinds of peptides are by causing synthetic maximum inhibition of glycogen in these receptor subtype physiology skeletal muscle.
Table 14
Calculate the EC of total glycogen content
50
The isolated rat musculus soleus of value and 95% confidence interval (CI) and In vitro culture
In
14
The C-conversion of glucose is the ratio of glycogen
Total glycogen content
14The C-conversion of glucose is a glycogen
(μ mol/g muscle dry weight/and h) (μ mol/g muscle dry weight/h)
-insulin+insulin-insulin+insulin
EC
50???????95％CI????EC
5???95％????EC
50???95％CI????EC
50???95％CI
0??????CI
Side chain 8.0 1.9-33.0,8. 3.5-1,9.4 0.2-476,9.2 4.0-21.0 that form sediment
CGRP-??????3.2?????????0.4-27.2??4.?????1.2-1???3.1?????0.3-27.8??0.7?????0.3-2.1
1????????????????????????????????3??????4.6
sCT????????1.7?????????0.01-48.??0.?????0.3-1???0.1?????0.1-52????0.9?????0.3-2.8
2?????????6??????.3
EC
50P=N.S.. p=N.S P=N.S. p＜0.01
Difference
Maximum p＜0.0001 p＜0.0001 p＜0.0001 p＜0.0001 that presses down
That makes is poor
Different
Lack and when having the insulin human (23.7nM) of maximum valid density, the rat soleus muscle bar is cultivated with amylopectin, CGRP and sCT, as shown in Figure 15.Determine significance by two-way ANOVA.
These tests show that having the metabolism amylopectin passed through calcitonin receptor (oppositely infix form) [C1 by the receptor of body characteristics
Ins-] and ramp 1 cotransfection in L6 sarcoplast or cos-7 cell, make up.These receptor foundation combine with the strong specificity of CGRP, amylopectin and sCT, and conduct by the signal of CGRP and amylopectin.On the contrary, rat calcitonin and people adrenomedullin, and the representative of CGRP/ amylopectin peptide family is lower to this receptor binding affinity.As shown in embodiment hereinafter, biological action and its effect in the isolated rat musculus soleus as part in recombinant receptor of these peptides compared, and musculus soleus is the known target tissue pharmaceutically of these peptides.The peptide part of being measured is to C1
Ins-The binding affinity of/ramp 1 receptor has and its representative EC to the synthetic inhibition of glycogen in complete skeletal muscle
50Be worth identical relative populations order.Agonist be in proper order sCT＞CGRP ≈ amylopectin＞＞rat calcitonin, people adrenomedullin.The similar EC that CGRP and amylopectin stimulate cAMP to obtain to the similar affinity of this receptor and these peptides
50Value is consistent.Tissue distribution studies show that in rat skeletal muscle, C1
Ins-Be to transcribe the main molecules that obtains from calcr.These results and CGRP and amylopectin in skeletal muscle as C1
Ins-/ ramp 1 receptor stimulating agent produces and suppresses consistent to glycogen is synthetic.Other CGRP receptor subtype is characterized on molecular level, by with ramp construction and or relevant G coupled receptor, calcitonin receptor sample receptor, CRLR cotransfection structure.See McLatchie etc., 1998, natural 393:333-339; With Buhlmann etc., 1999, endocrinology 140:2883-2890).Rat CGRP-1 and amylopectin also carry out the isolated rat musculus soleus of feeding from high lipid as the effect of insulin active antagonist.These tests show in the muscle that high lipid is fed, CGRP does not change as the effect of noncompetitive insulin active antagonist, and it is the not desired effects of glycogen to the conversion of glucose of insulin stimulating that the effect that lipid content in the muscle that high lipid is fed is described is different from antagonist.
Quick CGRP perfusion (1h) back is to organizing lipid and metabolic influence in the rat of normally feeding
The operation test is carried out on one's body at male Wistar rat.As described belowly randomly rat is divided in the test group.Induce and keep surgery anesthesia by 3-5% halothane and 2L oxygen.Take the initial blood sample to come free fatty, triglyceride and the cholesterol of the blood-glucose of establishment of base line level and lactic acid, electrolyte, soda acid and oxygen level, blood levels from arteria caudalis.After this, insert conduit immediately, feed to rat with 60-70 breathing/min and added O
2Air.Regulate respiratory frequency and hold morning and evening tides to press (end-tidal pressure) (10-15cmH to the greatest extent
2O) keep most end morning and evening tides CO
2At 35-40mmHg.In whole surgery and process of the test, make body temperature remain on 37 ℃ by heating platform.Carotid artery and jugular vein insert the solid phase pressure transducer respectively and add the PE50 urinary catheter of dress normal saline.For antagonist perfusion test, it is similar to the above to perform the operation, and the difference part is that carotid artery and jugular vein all insert the PE50 urinary catheter that adds the dress normal saline.Liquid (normal saline be dissolved in normal saline in hormone) be transported in the jugular vein with the Y-connection that is connected these two pipes.Carotid pipe is connected with pressure transducer.(base-line data), 30min and every 20min obtain blood sample up to the 90min of off-test by this pipe when zero.
PerfusionIn operation with at least behind the stable phase of 15min, with 1ml.h
-1Speed pour into normal saline (contrast) or CGRP (100pmol.kg to animal
-1.min
-1, Bachem) 60min.The additional speed intravenous injection 154mmol/L NaCl solution with 3.3ml/kg/h of this perfusion substitutes the liquid loss that has caused.During whole test, obtain the blood-glucose measured value with 5min interval (tail capillary) from afterbody capillary tube blood vessel.Perfusion obtains the terminal blood sample by cardiac puncture after finishing, and as each baseline sample analysis, and analyze various metabolites and hormone.Serum and plasma sample are preserved up to further analysis down at-80 ℃.Take off neck and put to death rat, collection organization's (lipid piece of brain, liver, kidney, skeletal muscle, extensor digitorum longus flesh, left ventricle, retroperitoneal and epididymis) is apace at N
2In freezing and preserve down up to further analysis at-80 ℃.For the antagonist test, method is same as described above, has following change: unite perfusion antagonist, CGRP and normal saline.Animal pro-30min accepts antagonist or normal saline with 1ml/kg/h, and then starts the second pipe that suitably contains CGRP or normal saline with 1ml/kg/h.
In entire test, monitor mean arterial pressure (MAP), heart rate (HR obtains), oxygen saturation (Nonin 8600V pulse oximeter and core body temperature constantly from the MAP waveform with the PowerLab/16s data acquisition module.Showing calibrating signal on the screen and be saved on the CD with the 2s meansigma methods of each variable.
The result
Perfusion CGRP produces the specific action to the lipid content in blood plasma, liver and the musculus soleusCompare with the control animal of perfusion normal saline, the CGRP that is 100pmol/kg/h to dopey animal perfusion dosage causes plasma C GRP to increase (6.3 ± 0.8 contrast ratios, 325 ± 75p; P＜0.01) (table 15).And the concentration of insulin, amylopectin, epinephrine and norepinephrine does not have significant change.
Table 15
Serum hormone concentration after the CGRP perfusion
Gather whole blood by cardiac puncture, quantize hormone with radioimmunoassay, RIA or HPLC.Numerical value is average ± SEM (each value is n=10 animal).By t-check analysis statistical significance.* is with respect to contrast significant difference, P＜0.01.
Serum hormone concentration (pM) contrast CGRP
Insulin 810 ± 103 631 ± 142
CGRP????????????????????????????6.3±0.8????????????????325±75**
Epinephrine 6776 ± 509 8321 ± 491
Norepinephrine 2194 ± 80 2734 ± 134
Compare with the perfusion normal saline, blood plasma free fatty acid concentration significantly raise (0.1mmol/L contrast ratio 0.4mmol/L, P＜0.01) (table 16) appears in perfusion CGRP.On the contrary, the CGRP antagonist, (CGRP-(8-37) causes the remarkable decline (0.1mmol/L contrast ratio-0.3, P＜0.05) of free fatty acid levels with the perfusion of the dosage of 10nmol/kg/h, shows that this antagonist can block the activity of endogenous CGRP peptide.In dabbling time course, CGRP causes that plasma triglyceride reduces (0.2mmol/L contrast ratio-0.7mmol/L, P0.01), and this antagonist shows opposite effect, the level of triglyceride is elevated to significant level (0.2mmol/L contrast ratio 0.9mmol/L, P＜0.01) in the filling process of 90min.Two kinds of peptides combination (CGRP-(8-37) 90min+CGRP 60min) does not cause the variation with respect to control level of blood plasma free fatty acid or content of triglyceride.
Table 16
Blood metaboilic level after the CGRP perfusion
Gather whole blood by cardiac puncture, with 3,000rpm is centrifugal 15min under 10 ℃, takes out serum and also preserves up to further analysis down at-80 ℃.Use the concentration that quantizes free fatty and triglyceride based on the method for enzyme.With the lactic acid in the whole blood of arterial blood gas analysis instrument mensuration heparnised.Shown in data be that metabolite concentration changes, the ratio of the metabolite concentration of the metabolite concentration after finishing with perfusion phase during divided by the perfusion beginning is represented (each point is n=7 animal).By t-check analysis statistical significance.With respect to contrast significant difference, * * P＜0.01; * P＜0.05.
[serum] changes (mmol/L)
Contrast CGRP CGRP-(8-37) CGRP-(8-37)+
CGRP
Ester
FFA???????????-0.1????????0.4**???????-0.3*??????????????-0.1
Lactic acid 0.3 1.7** 0.02 0.4
Serum hormone concentration (pM) normal saline is irritated the CGRP perfusion
Annotate
Insulin 810 ± 103 631 ± 142
CGRP????????????????????6.3±0.8??????????325±75**
Epinephrine 6776 ± 509 8321 ± 491
Norepinephrine 2194 ± 80 2734 ± 134
Lack or when having antagonist CGRP-(8-37), behind perfusion CGRP the lipid of tissue is analyzed, showing has specific effect (table 17) in skeletal muscle and liver.Significant change does not take place in triglyceride and free fatty acid content in heart, epididymis lipid (epidydmaladipose) or kidney.The CGRP perfusion causes the free fatty in skeletal muscle and the liver significantly to raise and content of triglyceride descends.On the contrary, only pour into antagonist these lipids are produced adverse effect, show that it has blocked the effect of endogenic CGRP.Behind common perfusion CGRP and the antagonist, lipid content does not have significant change (table 17).
Table 17
In skeletal muscle and liver, CGRP produces the tissue specificity effect to free fatty and triglyceride
After suitably using CGRP (100pmol/kg/min 1h), CGRP-(8-37) (10nmol/kg/min 90min), their combinations or normal saline perfusion 90min, the FFA (A) in various organs and the tissue and the content of triglyceride (B).The excision organ also adopts and determines total lipid content based on the method for enzyme.Numeric representation is average ± SEM (n=animal of each point).By single factor ANOVA analytical statistics significance, analyze with Dunnet multiple comparisons check carrying out post-hoc then.With respect to contrast significant difference, * P＜0.05; * P＜0.01; * * P＜0.001.
A
(μ mol/g) contrasts CGRP CGRP-(8-37) CGRP-(8-37)+CGRP
Heart 0.8+0.2 0.9+0.1 0.9+0.2 0.7+0.2
Liver 0.1+ 0.7+0.1** 0.06+0.04 0.1+0.01
0.01
Epididymis lipid 2.1+0.5 2.7+0.7 2.2+0.6 2.0+0.3
Musculus soleus 0.2+ 0.6+0.07** 0.1+0.02 0.2+0.04
0.02
Kidney 0.8+0.1 0.9+0.2 0.8+0.1 0.7+0.2
B
Heart 2.9+0.4 2.6+0.4 3.1+0.4 3.7+0.5
Liver 4.3+0.3 2.2+0.5** 5.8+0.4* 3.7+0.3
Epididymis lipid 5.1+1.2 5.0+0.9 4.9+1 5.2+1.3
Musculus soleus 3.7+0.4 1.5+0.2** 4.3+0.4* 3.6+0.5
Kidney 2.8+0.5 2.4+0.4 2.5+0.4 2.3+0.5
Embodiment 16
The CGRP perfusion dosage of 100pmol/kg/h is to carbohydrate metabolism and vasodilation generation effect
In the CGRP perfusion as embodiment 15 described 60min, mean arterial blood pressure reduces, and (accompanying drawing 20) in the end significantly descends among the 10min.Similarly, CGRP perfusion causes blood glucose concentration significantly raise (accompanying drawing 21).Compare with control animal, CGRP combines with its antagonist CGRP-(8-37) and also causes blood-glucose to raise.In CGRP perfusion, lactate level raises (0.3mmol/L to 1.7mmol/L, P＜0.01) with respect to control level, and this antagonist has adverse effect to its agonist, a little down with (0.3mmol/L contrast ratio 0.02, P＜0.05).Antagonist can be blocked the increase of the concentration of glucose that is caused by CGRP, and lactate level does not have respective change, and (the 0.3mmol/L normal saline is than 0.4mmol/L, P=NS).
Test among the embodiment 15 and 16 shows, realizes being enough to produce lactic acid and glucose generation, lipid metabolism and vasodilative effect with the dabbling CGRP dosage of 100pmol/kg/h.The CGRP-1 serum-concentration that occurs 300pM in the dabbling animal with 100pmol/kg/h is illustrated in the steady concentration in the perfusion phase of a few hours.(the removing half-life of supposing external CGRP-1 is 10min, and this concentration reaches behind 2-3 half-life or 20-30min).It is useful that the dosage that makes serum-concentration be lower than this does not act on metabolism amylopectin receptor for activating low affinity receptor.
Embodiment 17
The effect that causes by CGRP1 in the lipid oxygenase inhibitor Masoprocol counter-rotating musculus soleus to the muscle lipid
Masoprocol (nordihydroguaiaretic acid) is a kind of lipid oxygenase inhibitor, and being determined is the active component of creosote bush (Larrea divaricata) extract.Oral this chemical compound can reduce serum N EFA and triglyceride concentration in the rat of the inductive hypertriglyceridemia of fructose (Gowri etc. 2000, U.S. physiology magazine 279:E593-E600), with reduce lipid and feed/the streptozotocin rat model in blood-glucose and triglyceride concentration (Reed etc., 1999, diabetologist 42:102-106).Owing to shown and in the lipid cell, played lipotropism matter decomposition by inhibitory hormone sensitivity lipid enzyme by Masoprocol (Gowri etc. 2000, U.S. physiology magazine 279:E593-E600), studied it acts on NEFA and content of triglyceride to CGRP-1 in high lipid is fed the musculus soleus of rat influence.Separately cultivate the musculus soleus cutlet with 50 μ M masoprocol, ((3.9 ± 0.8nmol/g baseline does not act on than 4.5 ± 0.9mmol/g) (B) content is remarkable 74 ± 30nmol/g baseline to 55.77 ± 21.08mmol/g) (A) or triglyceride to NEFA.But masoprocol reverses fully by 100nM CGRP 1 (533 ± 114nmol/g; P＜0.001) and 1pMCGRP 1 (482 ± 99nmol/g; P＜0.05) NEFA that causes of nmol/g raises.Similarly, masoprocol reverses fully by 100nM CGRP 1 (1.2mmol/g ± 0.3nmol/g; P＜0.05) and 1pM CGRP 1 (1.2mmol/g ± 0.1nmol/g; P＜0.05) causes the decline of content of triglyceride in the musculus soleus.
Be to be understood that embodiment described herein and embodiment only are used for illustration purpose; and according to the various improvement of these contents or change and instructed to those skilled in the art, be included in the protection domain of the application's spirit and scope and additional claim.All publications, patent, patent application and the accession number that this paper quotes (comprising many application and/or nucleotide and peptide sequence and corresponding note the in first to file before the applying date) is incorporated herein by reference in full at this, has with each publication, patent or patent application so to be introduced into identical as a reference purpose especially and respectively.
Claims (31)
1. determine whether a kind of reagent is used in the method that mammal moderate stimulation lipid decomposes, comprise and determine whether this reagent is the agonist of high-affinity CGRP receptor.
2. the method for claim 1 comprises that further definite this reagent preferably stimulates high-affinity CGRP receptor with respect to metabolism amylopectin receptor.
3. the method for claim 2 comprises that further this reagent relatively is used to influence the EC by the receptor-mediated reaction of metabolism amylopectin
50Be used to influence EC with this reagent by the receptor-mediated reaction of high-affinity CGRP
50
4. the method for claim 3 is wherein used the external definite EC of skeletal muscle that exsomatizes
50Value.
5. the method for claim 3, wherein the reaction by the high-affinity receptor mediation is that free fatty acid increases in the skeletal muscle tissue, is influence to carbohydrate metabolism by the reaction of metabolism amylopectin mediation.
6. determine the whether method of useful as therapeutics of a kind of reagent, comprising:
Determine ii) whether a kind of reagent decomposes at the mammalian tissues moderate stimulation lipid of expressing high-affinity CGRP receptor and metabolism amylopectin receptor; With,
Iiii) determine a kind of from i) reagent whether preferably stimulate high-affinity CGRP receptor with respect to metabolism amylopectin receptor,
Wherein preferably stimulate the reagent of high-affinity CGRP receptor to be defined as useful as therapeutics.
7. the method for claim 6, wherein this tissue is a skeletal muscle.
8. determine whether a kind of reagent is used in the method that mammal moderate stimulation lipid decomposes, comprise relatively the lipid degrading activity of this reagent and the lipid degrading activity of CGRP.
9. determine whether a kind of reagent is used in the method that mammal moderate stimulation lipid decomposes, comprise relatively the lipid degrading activity of this reagent and the lipid degrading activity of CGRP.
10. determine the dosage of agonist of high-affinity CGRP receptor and the method for prescription, it does not cause the side effect of not expecting in the decomposition of mammalian bone flesh moderate stimulation lipid in this mammal, the described side effect of not expecting is that blood-glucose, blood lactic acid raise or vasodilation, comprises
Ii) pass through a) to give a large amount of various dose of administration of test or the CGRP receptor stimulating agent of prescription;
B) measure the influence that each dosage or prescription decompose the lipid of testing in the mammiferous tissue and measure various dosage the dose response analysis is carried out in the influence of side effect, lipid decomposes and the dose response data of side effect thereby set up; With,
Iiii) from the dose response data, determine to stimulate lipid to decompose and do not cause the dosage of the CGRP receptor agonism agent prescription of side effect.
11. the method for claim 10, wherein various dosage or prescription are determined by the free fatty acid levels of measuring in the animal tissue the influence that lipid decomposes.
12. one kind is used to be administered to mammiferous pharmaceutical composition in unit dose form, described unit dose contains is enough in blood to produce the high-affinity CGRP receptor stimulating agent and the pharmaceutically acceptable excipient of amount that preferably stimulates the agonist level of high-affinity receptor with respect to metabolism amylopectin receptor.
13. the pharmaceutical composition in unit dose form of claim 12, wherein this agonist is CGRP-1 or its functional variant biologically.
14. the pharmaceutical composition in unit dose form of claim 12, wherein this agonist is that the content of agonist in CGRP-1 and the blood is lower than 300pM.
15. the pharmaceutical composition in unit dose form of claim 12, wherein this agonist is that the content of agonist in CGRP-1 and the blood is about 10
-15M is to about 10
-10Between the M.
16.CGRP be used for the treatment of in preparation and suffer from or susceptible is characterized as purposes in the mammiferous medicine of symptom of accumulation of lipid in the skeletal muscle, wherein when being administered to mammal, this medicine produces the agonist level that is lower than 300pM in blood.
17. the purposes of claim 16, wherein this symptom is diabetes, insulin resistant or X syndrome.
18.CGRP be used for the treatment of in preparation and suffer from or susceptible is characterized as purposes in the mammiferous medicine of symptom of accumulation of lipid in the skeletal muscle, wherein when being administered to mammal, this medicine is created in about 10 in blood
-15M is to about 10
-10Agonist level between the M.
19. being used for the treatment of in preparation, high-affinity CGRP receptor stimulating agent suffers from or susceptible is characterized as purposes in the mammiferous medicine of symptom of accumulation of lipid in the skeletal muscle, wherein when being administered to mammal, this medicine produces the agonist level that is enough to preferably stimulate with respect to metabolism amylopectin receptor high-affinity receptor in mammal.
20. the purposes of claim 19, wherein this agonist is CGRP-1.
21. the method exsomatizing mammalian cell and organizing the moderate stimulation lipid to decompose comprises cell or tissue is contacted with high-affinity CGRP receptor and/or metabolism amylopectin receptor stimulating agent, and measures the variation of lipid decomposition level in this tissue.
22. the method for claim 21 wherein detects the variation of lipid decomposition level with the variation of free fatty amount.
23. in the method that exsomatizes mammalian cell and organize the moderate stimulation lipid to decompose, comprise cell or tissue is contacted with high-affinity CGRP receptor stimulating agent, wherein this tissue is from skeletal muscle or liver, and wherein this tissue be enough to preferably to stimulate the agonist of the amount of high-affinity CGRP receptor to contact with respect to metabolism amylopectin receptor.
24. the method for claim 23, wherein this agonist is CGRP-1.
25. the method for claim 24, wherein the amount of CGRP-1 is about 10
-15M is to about 10
-10Between the M.
26. the method for claim 23 further comprises the step that detects the stimulation of in the tissue lipid being decomposed.
27. in exsomatize mammalian bone flesh or liver, suppress the method that lipid decomposes, comprise skeletal muscle or liver metabolism amylopectin receptor and/or the high-affinity CGRP receptor antagonist with the amount that is enough to suppress the lipid decomposition contacted.
28. the method for claim 27, wherein antagonist is
8,37Amylopectin or
8,37CGRP.
29. the method in that mammalian bone flesh moderate stimulation lipid decomposes comprises this tissue is contacted with metabolism amylopectin receptor stimulating agent.
30. the method for claim 29, wherein agonist is CGRP.
31. a therapeutic scheme comprises that (i) gives and to suffer from or susceptible is characterized as administration CGRP-1 polypeptide, its biological function variant or its metabolism receptor for stimulating variant of the symptom of accumulation of lipid in the skeletal muscle and the lipid (ii) monitored in this mammal decomposes.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US33342201P | 2001-11-26 | 2001-11-26 | |
| US60/333,422 | 2001-11-26 | ||
| NZ515731 | 2001-11-26 | ||
| NZ51573101 | 2001-11-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1617737A true CN1617737A (en) | 2005-05-18 |
Family
ID=26652295
Family Applications (1)
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|---|---|---|---|
| CNA028275691A Pending CN1617737A (en) | 2001-11-26 | 2002-11-26 | Compositions and method for normalizing lipid levels in mammalian tissues |
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| EP (1) | EP1461068A4 (en) |
| JP (1) | JP2005523418A (en) |
| CN (1) | CN1617737A (en) |
| AU (2) | AU2002356469A1 (en) |
| CA (1) | CA2471833A1 (en) |
| WO (1) | WO2003045424A1 (en) |
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|---|---|---|---|---|
| WO2000018392A1 (en) | 1998-09-25 | 2000-04-06 | Glycox Corporation Limited | Fructosamine oxidase: antagonists and inhibitors |
| ES2386782T3 (en) | 2002-03-08 | 2012-08-30 | Philera New Zealand Limited | Prevention and / or treatment of cardiovascular disease and / or associated heart failure |
| EP1539129A4 (en) | 2002-08-20 | 2006-03-08 | Protemix Corp Ltd | Dosage forms and related therapies |
| DK1778618T3 (en) | 2004-07-19 | 2014-03-10 | Philera New Zealand Ltd | Synthesis of triethylenetetramines |
| US8168592B2 (en) | 2005-10-21 | 2012-05-01 | Amgen Inc. | CGRP peptide antagonists and conjugates |
| WO2012162243A2 (en) | 2011-05-20 | 2012-11-29 | Alderbio Holdings Llc | Anti-cgrp compositions and use thereof |
| MX356269B (en) | 2011-05-20 | 2018-05-21 | Alderbio Holdings Llc | Use of anti-cgrp antibodies and antibody fragments to prevent or inhibit photophobia or light aversion in subjects in need thereof, especially migraine sufferers. |
| CN103957935B (en) | 2011-05-20 | 2018-04-03 | 奥尔德生物控股有限责任公司 | Anti- CGRP or anti-CGRP R antibody or antibody fragment are used to treat or prevent the chronic and diarrhoea of acute form purposes |
| SG11201510618YA (en) * | 2013-07-03 | 2016-03-30 | Alder Biopharmaceuticals Inc | Regulation of glucose metabolism using anti-cgrp antibodies |
| CR20210373A (en) | 2019-01-08 | 2021-08-19 | H Lundbeck As | Acute treatment and rapid treatment of headache using anti-cgrp antibodies |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8720115D0 (en) * | 1987-08-26 | 1987-09-30 | Cooper G J S | Treatment of diabetes mellitus |
| US5175145A (en) * | 1988-08-26 | 1992-12-29 | Amylin Pharmaceuticals, Inc. | Treatment of diabetes mellitus with amylin agonists |
| WO1994021665A1 (en) * | 1993-03-24 | 1994-09-29 | Amylin Pharmaceuticals, Inc. | Cloned receptors and methods for screening |
| WO1996005221A1 (en) * | 1994-08-16 | 1996-02-22 | Human Genome Sciences, Inc. | Calcitonin receptor |
| WO1998003534A1 (en) * | 1996-07-23 | 1998-01-29 | Smithkline Beecham Corporation | Calcitonin gene-related peptide receptor component factor (houdc44) |
| CN1055640C (en) | 1996-11-29 | 2000-08-23 | 沃维汉 | Human calcium degrading gene concerned peptide fatty composite and preparation thereof |
| ES2356595T3 (en) * | 1998-02-13 | 2011-04-11 | Amylin Pharmaceuticals, Inc. | NEW MIXED ACTIVITY COMPOUNDS OF THE AMILINA. |
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2002
- 2002-11-26 CA CA002471833A patent/CA2471833A1/en not_active Abandoned
- 2002-11-26 CN CNA028275691A patent/CN1617737A/en active Pending
- 2002-11-26 EP EP02803945A patent/EP1461068A4/en not_active Withdrawn
- 2002-11-26 AU AU2002356469A patent/AU2002356469A1/en not_active Withdrawn
- 2002-11-26 WO PCT/NZ2002/000262 patent/WO2003045424A1/en active Application Filing
- 2002-11-26 JP JP2003546925A patent/JP2005523418A/en active Pending
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2009
- 2009-03-26 AU AU2009201147A patent/AU2009201147A1/en not_active Abandoned
Also Published As
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|---|---|
| CA2471833A1 (en) | 2003-06-05 |
| EP1461068A1 (en) | 2004-09-29 |
| WO2003045424A1 (en) | 2003-06-05 |
| JP2005523418A (en) | 2005-08-04 |
| AU2009201147A1 (en) | 2009-04-23 |
| EP1461068A4 (en) | 2006-03-29 |
| AU2002356469A2 (en) | 2003-06-10 |
| AU2002356469A1 (en) | 2003-06-10 |
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