CN1980954A - Amylin family peptides and methods for making and using them - Google Patents

Abstract

The present invention relates to novel compounds having a function of a peptide in the amylin family, related nucleic acids, expression constructs, host cells, and processes production of the compounds. The compounds of the invention include one or more amino acid sequence modifications. In addition, methods and compositions are disclosed to treat and prevent metabolic disorders such as obesity, diabetes, and increased cardiovascular risk.

Description

Amylin family peptides and preparation thereof and using method

Related application

The present invention requires the right of priority of No. the 60/543rd, 275, the U.S. Provisional Application of submitting on February 11st, 2004 and the U.S. Provisional Application of submitting on March 3rd, 2004 the 60/550th, No. 447, and these two provisional application all full text are hereby incorporated by.

Technical field

The present invention relates to novel cpd and preparation thereof and using method.These compounds can be used for treating or prevent Metabolic disorder, vascular disease, ephrosis and or the illness of gastrointestinal illness and so on.Representative illness can be the valuable illness of minimizing of wherein ingestion of food or calorie intake, for example obesity, type ii diabetes, eating disorder, metabolic syndrome and insuline resistance syndrome.

Background of invention

Up to now, for understanding the 26S Proteasome Structure and Function of islet amyloid polypeptide, thyrocalcitonin and calcitonin-gene-related peptide (CGRP), it many work have been carried out.Table 1 provides the general introduction of some biological effects of CGRP, thyrocalcitonin and islet amyloid polypeptide, and this general introduction is disclosed in Wimalawansa, S.J. (1997) Critical Reviews in Neurobiology, 11; 167-239.

Table 1:

Biological effect	CGRP	Thyrocalcitonin	Islet amyloid polypeptide
				Vasorelaxation	++++	+/-	++
Chronotropic effect	++	-	++
				Inotropic effect	++	-	++
Suppress bone resorption	+	++++	++
				Stimulate bone forming	-	-	+
The calcium effect is fallen	-	+++	++
				The effect of long factor sample	+	-	+

Neurotization	++	+/-	-
				Thermoregulation	+	-	+
Inflammation	++	+/-	-
				Gastric mucosal protection	++	-	-
Effect to the β cell	+	+/-	++
				Skeletal muscle	+	-	+
Glucose metabolism	+	-	+
				The appetite stimulator effect	+	++	++
Analgesic activity	-	++	+

Reported already that islet amyloid polypeptide was regulated stomach emptying, therefore glucagon suppression secretion and ingestion of food regulate the glucose generating rate in the circulation.It seems the supplementation with insulin effect, and Regular Insulin is regulated the absorption to glucose of glucose clearance speed in the circulation and peripheral tissues.These effects obtain the support of the experimental result in rodent and people, and experimental result shows islet amyloid polypeptide with the effect of at least three kinds of independent mechanism supplementation with insulin in GLPP control, and all these mechanism are the affecting glucose generating rate all.At first, islet amyloid polypeptide suppresses glucagon secretion after the meal.Compare with health adult, the type 1 diabetes patient does not have the circulation islet amyloid polypeptide, and diabetes B patient islet amyloid polypeptide concentration after the meal reduces.And infusion causes the contrast of hyperglycemic-glycogenolytic factor concentration ratio that very big lifting is arranged again in conjunction with the islet amyloid polypeptide monoclonal antibody specific of circulation islet amyloid polypeptide.These two results have pointed out the physiological action of endogenous islet amyloid polypeptide in glucagon secretion is regulated after the meal.Secondly, slow down digestive tract power and stomach emptying of islet amyloid polypeptide.At last, inferior colliculus intracerebral injection rat Langerhans islet amyloid polypeptide demonstrates the reduction rat and gets food, changes the neurotransmitter metabolism in the hypothalamus.In some research, behind inferior colliculus intracerebral injection rat Langerhans islet amyloid polypeptide and rat CGRP, ingestion of food significantly reduction reaches 8 hours.In human experimentation, islet amyloid polypeptide analogue tripro-amylin has shown the increase that reduces body weight or body weight.Islet amyloid polypeptide has the treatment of benefiting metabolic disorder, for example diabetes and obesity.Islet amyloid polypeptide also can be used for treating pain, osteopathia, gastritis, is used to regulate lipid (particularly triglyceride level), or is used to influence organization, and for example fat preferentially reduces and saving lean tissue mass.

Hormone thyrocalcitonin (CT) is secreted under the effect of inductive hypercalcemia because of it and is reduced the blood calcium effect fast and gain the name.Produce in the Tiroidina neuroendocrine cell of its former C of called after cell, and by this emiocytosis.The CT that studies at most (1-32) effect is its effect to osteoclast.The interaction in vitro of CT comprises that ruffed border disappears fast and lysosomal enzyme discharges decline.At last, CT causes bone resorption to descend to the inhibition of osteoclast function.But the change of serum C T that exists in chronic minimizing of the change of serum C T under the thyroidectomy situation and the medullary substance thyroid carcinoma increases, and seems all irrelevant with the variation of serum calcium or sclerotin.Therefore, most possible is, the major function of CT (1-32) is in case of emergency to resist acute hypercalcemia and/or at " calcium stress " phase (for example vegetative period, the Gestation period and lactation) protection bone.(summarizing in Becker JCEM, 89 (4): 1512-1525 (2004) and Sexton, Current MedicinalChemistry 6:1067-1093 (1999)).Corresponding to therewith is the data that obtained by the calcitonin gene knock-out mice of having removed thyrocalcitonin and CGRP-I peptide simultaneously in the recent period, data disclose the basic calcium correlation that mouse has normal level, but blood calcium reaction increases (Kurihara H etc., Hypertens Res.2003 February; 26 Suppl:S105-8).

CT has effect to the plasma calcium level, suppresses the osteoclast function, is widely used in the treatment osteoporosis.From treatment, salmon CT seems bone density improving, reduces fracture rates, and the side effect minimum.In in the past 25 years, CT also has been successfully used to treat bone Paget disease, and bone Paget disease is a kind of chronic skeletal diseases, can cause the bone in one or more bony areas to become big or distortion.CT also is widely used owing to its analgesic activity to the ostalgia experienced in the osteoporosis process, although the mechanism of this effect of not clear understanding.

Calcitonin-gene-related peptide (CGRP) is that a kind of its acceptor is distributed widely in the neuropeptide of (comprising neural system and cardiovascular systems) in the body.As if this peptide is regulated the sensory nerve transmission, is one of the most effective endogenous vasorelaxation peptide of finding up to now.The CGRP biological effect of having reported comprises: regulate P material in the inflammation, regulate neuromuscular junction the nicotine receptor active, stimulate the pancreatin secretion, reduce gastric acid secretion, peripheral vasodilation, palpitate quickly, neuroregulation, Calcium Metabolism Regulation, skeletonization stimulation, insulin secretion, body temperature increases and ingestion of food reduces.(Wimalawansa，Amylin，calcitonin gene-related peptide，calcitonin and ADM：a peptide superfamily.Crit Rev Neurobiol.1997；11(2-3)：167-239)。The vital role of CGRP is to utilize its effective vasorelaxation action control to lead to the blood flow of each organ, give CGRP with vein after mean arterial pressure drop to card.The vasorelaxation action support that the CGRP knock-out mice is analyzed recently that also obtains isozygotying, the sympathetic activity increase causes that peripheral vascular resistance improves and hypertension (Kurihara H etc., Targeted disruption of ADM and alpha CGRP genesreveals their distinct biological roles.Hypertens Res.2003 February around this analysis revealed; 26Suppl:S105-8).Therefore, as if except other effect, CGRP also excites vasorelaxation action, hypotensive activity and increase heart rate.

Patients with congestive heart failure long period infusion CGRP has been shown lasting beneficial effect to the Hemodynamics function, and be free from side effects, prompting CGRP can be used for heart failure.Other indication that CGRP uses comprises renal failure, acute and chronic coronary ischemia, treatment heart disorder, other peripheral vascular disease, for example Raynaud phenomenon, subarachnoid hemorrhage, hypertension and pulmonary hypertension.Also can treat the PET of gestation and premature labor potentially.(Wimalawansa，1997)。Recent treatment is used and is comprised use CGRP antagonist for treating migraine.

The various peptides that existence can be used for the treatment of alone or in combination or prevent various illnesss, has many beneficial characteristics.New and the useful peptide that establishment has multiple effect should be useful, and the improved characteristics that existing peptide does not have is given in wherein said multiple effect.For example, in ingestion of food experiment, shown islet amyloid polypeptide fast-acting in 30 minutes, but it acts on after 60 minutes and stops gradually.On the contrary, shown that salmon calcitonin see calcimar has delayed action, peak level still kept in the time of 240 minutes.Can simulate the effect of islet amyloid polypeptide and/or thyrocalcitonin and have the quick outbreak activity that is similar to islet amyloid polypeptide and this novel cpd of the continuous activity of thyrocalcitonin, can increase the ability and the effect of any independent compound.And some physicochemical characteristic of islet amyloid polypeptide, thyrocalcitonin and/or CGRP is combined into single form, and the different positions that can be beneficial in the dysfunction metabolic cycles disturbs.These novel cpds are the required activity or the property combination of good therapeutical agent, can produce and have at least a required feature the compound of (for example higher effect, high ability, higher bioavailability, side effect less, be easy to production, stability and/or solvability).

The All Files that this paper mentions all is attached among the application by reference, sets forth fully as this paper.

Summary of the invention

The present invention relates to novel amylin family peptides or compound at least in part.Aspect general, these novel cpds (being also referred to as The compounds of this invention) have: the district of ring at least of islet amyloid polypeptide or thyrocalcitonin and analogue thereof; The alpha helical region of at least a portion thyrocalcitonin or its analogue alpha helical region, or have the alpha helical region of part islet amyloid polypeptide alpha helical region and thyrocalcitonin alpha helical region or its analogue separately; And the C of islet amyloid polypeptide or thyrocalcitonin or its analogue end tail, precondition is that the C end tail of thyrocalcitonin or calcitonin-like is not proline(Pro) (Pro), oxyproline (Hyp), homoserine (Hse) or Hse derivative.

" analogue " used herein refers to that its sequence derives from the peptide of basic reference peptide (for example islet amyloid polypeptide and thyrocalcitonin) sequence, the insertion, displacement, extension and/or the disappearance that comprise the reference aminoacid sequence, preferably have at least 50% or 55% amino acid sequence identity, more preferably have at least 70%, 80%, 90% or 95% amino acid sequence identity with basic peptide with basic peptide.In one embodiment, such analogue can comprise conservative or non-conservative amino acid replacement (comprising alpha-non-natural amino acid and L and D form).Analogue comprises compound with agonist activity and the compound with antagonistic activity.Analogue as defined herein also comprises derivative.

" derivative " is defined as: aforesaid reference peptide or analogue, its one or more amino acid side groups, alpha-carbon atom, terminal amino group or terminal carboxyl(group) have chemically modified.Chemically modified includes but not limited to add chemical part, creates new key and removes chemical part.Amino acid side group is modified and is included but not limited to Methionin epsilon-amino acidylate; Arginine, Histidine or Methionin N-alkylation; L-glutamic acid or aspartic acid carboxyalkylization; And glutamine or l-asparagine desamidization.Terminal amino group is modified and is included but not limited to deaminizating, N-low alkyl group, N-two-low alkyl group and the modification of N-acyl group.Terminal amino group is modified and is included but not limited to deaminizating, N-low alkyl group, N-two-low alkyl group and the modification of N-acyl group, for example alkyl acyl, branch's alkyl acyl, alkylaryl-acyl group.Terminal carboxyl(group) is modified and is included but not limited to acid amides, low alkyl group acid amides, dialkyl amide, arylamide, alkylaryl acid amides and lower alkyl esters modification.Low alkyl group is the C1-C4 alkyl.And, can utilize the known protecting group of general synthesising chemical technology personnel to protect one or more side groups or end group.But amino acid whose alpha-carbon monomethylization or dimethylization.

In certain embodiments, novel cpd has at least a portion and the islet amyloid polypeptide or the islet amyloid polypeptide analogue C end tail of islet amyloid polypeptide or islet amyloid polypeptide analogue ring district, thyrocalcitonin or calcitonin-like alpha helical region.In other embodiments, novel cpd has at least a portion and the islet amyloid polypeptide or the islet amyloid polypeptide analogue C end tail of thyrocalcitonin or calcitonin-like ring district, thyrocalcitonin or calcitonin-like alpha helical region.In other embodiments, novel cpd has at least a portion of islet amyloid polypeptide or islet amyloid polypeptide analogue ring district, islet amyloid polypeptide or islet amyloid polypeptide analogue alpha helical region and at least a portion of thyrocalcitonin or calcitonin-like alpha helical region, and islet amyloid polypeptide or islet amyloid polypeptide analogue C end tail.In other embodiments, novel cpd has at least a portion of thyrocalcitonin or calcitonin-like ring district, islet amyloid polypeptide or islet amyloid polypeptide analogue alpha helical region and at least a portion of thyrocalcitonin or calcitonin-like alpha helical region, and islet amyloid polypeptide or islet amyloid polypeptide analogue C end tail.In other embodiments, novel cpd has a part or at least a portion of islet amyloid polypeptide or islet amyloid polypeptide analogue alpha helical region and at least a portion of thyrocalcitonin or calcitonin-like alpha helical region of islet amyloid polypeptide or islet amyloid polypeptide analogue ring district, thyrocalcitonin or calcitonin-like alpha helical region, and thyrocalcitonin or calcitonin-like C end tail.

In certain embodiments, the ring district of novel cpd can further comprise in islet amyloid polypeptide or thyrocalcitonin ring and analogue thereof and is no more than 1,2,3 or 4 modification, comprises displacement, inserts or disappearance.Further the imagination novel cpd can have other modification containing the N end parts that N-adds the ring in cap district; it can have hydrophobic or hydrophilic feature; for example ethanoyl, dissident's acyl group, 3; 6;-dioxy base is sad or 1-is amino-4; 7,10-trioxa-13-tridecyl amine succinamic acid (succinimic acid).Modification can further comprise 1,2,3 or more a plurality of other amino acid.This is a zone that allows a plurality of modifications, can't address too much to such an extent as to modify, but those skilled in the art will appreciate that this point based on the modification that further exemplifies among the application.

Generally speaking, with regard to aminoacid sequence, term " modification " comprises displacement, insertion, prolongation, disappearance and derivation alone or in combination.Novel cpd of the present invention can comprise the modification of one or more " non-essential amino acid " residue.Under background of the present invention, the residue of the agonist activity of analogue polypeptide is not eliminated or do not reduced substantially to " nonessential " amino-acid residue for can change (i.e. disappearance or displacement) in the novel amino acid sequence.

Displacement comprises conservative amino acid replacement." conservative amino acid replacement " is that wherein amino-acid residue is had the displacement that the amino-acid residue of similar side chain or physicochemical characteristic (for example static, hydrogen bonding, etc. structure, hydrophobic character) replaces.Amino acid can be natural or alpha-non-natural amino acid.Amino-acid residue classification with similar side chain is known in the art.These classifications comprise amino acid with basic side chain (Methionin for example, arginine, Histidine), amino acid (aspartic acid for example with acid side-chain, L-glutamic acid), amino acid (glycine for example with uncharged polar side chain, l-asparagine, glutamine, Serine, Threonine, tyrosine, methionine(Met), halfcystine), amino acid (L-Ala for example with non-polar sidechain, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, tryptophane), amino acid (Threonine for example with β-branched building block, Xie Ansuan, Isoleucine) and have the amino acid (tyrosine for example of aromatic series side chain, phenylalanine, tryptophane, Histidine).Displacement also can comprise non-conservative change.

Compound of the present invention also can be by further derivation of chemically changed (for example amidation, glycosylation, acidylate, sulfation, phosphorylation, acetylize and cyclisation).Such chemically changed can and be passed through method or the acquisition of its arbitrary combination in the body by chemistry or biochemical method.The derivative of The compounds of this invention also can comprise puts together one or more polymkeric substance or small molecules substituting group.It is N end or C end or the amino-acid residue side chain that the fatty acid chain of polyoxyethylene glycol (" PEG ") polymkeric substance, polyamino acid (for example polyhistidyl, poly arginine, polylysine etc.) and/or all lengths is connected or is attached to the AFP-6 analogue that one base polymer is puted together.The small molecules substituting group comprise short alkyl and limited alkyl (constrained alkyl) (for example side chain, ring-type, condense, adamantyl) and aryl.In addition, alkaline residue such as R or K can be replaced by high R and high K, citrulline or ornithine, to improve the metabolic stability of peptide.The compounds of this invention also comprises the peptide of acid and acid amides form.

In certain embodiments, the alpha helical region of novel cpd comprises at least 4 continuous amino acids of thyrocalcitonin or calcitonin-like alpha helical region.In other embodiments, alpha helical region comprises at least 5,6,7 or 8 continuous amino acids of thyrocalcitonin or calcitonin-like alpha helical region.In other embodiments, alpha helical region comprises at least 10,11,12,13,14,15,16,17,18,19,20,21 or more a plurality of continuous amino acid of thyrocalcitonin or calcitonin-like alpha helical region.In certain embodiments, when the number of continuous amino acid less than 8 the time, the imagination alpha helical region further comprises at least 4,5,6,7,9,10,11 or more a plurality of continuous amino acid of islet amyloid polypeptide or islet amyloid polypeptide analogue alpha helical region.In certain embodiments, imagination can exist less thyrocalcitonin or calcitonin-like amino acid, more islet amyloid polypeptide or islet amyloid polypeptide analogue amino acid in the alpha helical region of novel cpd.The amino acid no that constitutes alpha helical region can be about 10 to 23 amino acid.Therefore, alpha helical region can be 10,11,12,13,14,15,16,17,18,19,20,21,22 or 23 amino acid longs.And described amino acid should provide about 3 to about 6 α spiral corners.The alpha helical region of also imagining novel cpd can further comprise in the alpha helical region of thyrocalcitonin and/or islet amyloid polypeptide alpha helical region and analogue thereof and is no more than 1,2,3,4,5,6,7,8 or 10 modification, comprises displacement, inserts or disappearance.

In certain embodiments, the C of novel cpd end tail comprises at least 6,5 or 4 amino acid of islet amyloid polypeptide or thyrocalcitonin and analogue thereof.In certain embodiments, the C of novel cpd end tail comprises the C end tail that at least a portion has βZhuan Jiao.In certain embodiments, introduce βZhuan Jiao by the combination of Gly-Ser amino acid.Therefore, novel cpd can have such C end tail, and promptly it comprises the islet amyloid polypeptide that has Gly-Ser or begin with Gly-Ser or the part (and analogue) of thyrocalcitonin C end tail.

In certain embodiments, the C of novel cpd holds tail further to comprise in islet amyloid polypeptide or thyrocalcitonin ring and analogue thereof and is no more than 1,2 or 3 modification, comprises displacement, inserts or disappearance.Further the imagination novel cpd can hold the C end parts of tail to have other modification at C, it can comprise L-octyl group glycine, 4ABU (4-aminobutyric acid), 9Anc (9 aminononanoic acid), 3,6-dioxy base is sad or 1-is amino-4,7,10-trioxa-13-tridecyl amine succinamic acid.Modification can further comprise 1,2,3 or more a plurality of other amino acid.The modification that those skilled in the art further exemplify according to the present invention should be understood the imagination modified types in this zone.

In one aspect, the ring district is defined as being present in 5-8 the amino acid whose zone at least that contain of N-end, wherein first amino acid and last amino acid can produce key, for example residue of the residue of islet amyloid polypeptide 2-7 position or thyrocalcitonin 1-7 position and its corresponding zone in the analogue separately thereof.On the other hand, alpha helical region is defined as in islet amyloid polypeptide or the thyrocalcitonin holding tail, structurally forming the internal portion of α spiral in abutting connection with ring district and C, for example residue of the residue of islet amyloid polypeptide 8-23 position or thyrocalcitonin 8-27 position and its corresponding zone in the analogue separately thereof.More on the one hand, C end tail is defined as the zone after the α spiral, for example residue of islet amyloid polypeptide 33-37 position or longer residue, for example residue of 27-37 position, or thyrocalcitonin 27 or 28 to 32 s' residue.The compounds of this invention had both comprised the acid amides form of disclosed compound, also comprised its sour form.

The islet amyloid polypeptide of this paper definition and thyrocalcitonin comprise all natural variants and plant variant.The example of islet amyloid polypeptide and thyrocalcitonin includes but not limited to:

People's islet amyloid polypeptide (hAmylin)

KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQID NO：1)

Rat Langerhans islet amyloid polypeptide (rAmylin)

KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY (SEQID NO：2)

Salmon calcitonin see calcimar (sCT)

CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP(SEQ ID NO：3)

Human calcitonin (hCT)

CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (SEQ IDNO：4)。

So-called " amino acid " and " amino-acid residue " refers to the amino acid of natural amino acid, alpha-non-natural amino acid and modification.Unless regulation on the contrary, general or all comprise D and L steric isomer with any amino acid that title is specifically noted, if its structure allows such stereoisomer form.Natural amino acid comprises L-Ala (Ala), arginine (Arg), l-asparagine (Asn), aspartic acid (Asp), halfcystine (Cys), glutamine (Gln), L-glutamic acid (Glu), glycine (Gly), Histidine (His), Isoleucine (Ile), leucine (Leu), Methionin (Lys), methionine(Met) (Met), phenylalanine (Phe), proline(Pro) (Pro), Serine (Ser), Threonine (Thr), tryptophane (Trp), tyrosine (Tyr) and Xie Ansuan (Val).Alpha-non-natural amino acid includes but not limited to high-lysine, homoarginine, homoserine, the azetidine carboxylic acid, the 2-aminoadipic acid, the 3-aminoadipic acid, Beta-alanine, alanine, the 2-aminobutyric acid, the 4-aminobutyric acid, 6-aminocaprolc acid, the 2-aminoheptylic acid, the 2-aminoisobutyric acid, the 3-aminoisobutyric acid, the 2-diaminopimelic acid, tertiary butyl glycine, 2,4-diamino isopropylformic acid, desmosine, 2,2 '-diaminopimelic acid, 2, the 3-diaminopropionic acid, Ethylglycocoll, the N-ethyl asparagine, high proline(Pro), hydroxylysine, other hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, isodesmosine, not-Isoleucine, the N-methylalanine, sarcosine, N-methyl Isoleucine, N-methyl amyl glycine, the N-methylvaline, naphthylalanine, norvaline, nor-leucine, ornithine, the amyl group glycine, 2-pyridine acid and Thioproline.Other alpha-non-natural amino acid is included on amino or its side-chain radical of its N end reversible or irreversibly sealed by chemistry or by the modified amino acid residue of chemically modified, and for example wherein side chain functionalities is methylate D and L amino acid or a residue of the N-of another kind of functional group by chemically modified.For example, the amino acid of modification comprises methionine sulfoxide, methionine(Met) sulfone; A kind of modified amino acid-aspartic acid of aspartic acid-(β-methyl esters); A kind of modified amino acid-Ethylglycocoll of glycine; Or a kind of modified amino acid-L-Ala methane amide of L-Ala.Other residue that can add is described in Sandberg etc., J.Med.Chem.41:2481-91,1998.

Aspect general, compound of the present invention comprises and encircles district, alpha helical region and C end tail at least.Ring district comprises the amino sequence that contains formula X-Xaa1 sequence-Y, and wherein X and Y can produce key, and is the independent residue of selecting with following side chain: thus described side chain each other chemical bonding form intramolecularly and connect, disulfide linkage for example; Amido linkage; Can form the alkyl acid and the alkylamine of cyclic lactames; Alkyl aldehydes or alkylogen and alkylamine, but its condensation and being reduced, thus alkylamine or imine linkage formed; Or thereby described side chain can connect formation alkyl, thiazolinyl, alkynyl, ether or thioether bond.Alkyl chain can comprise having about 1 low alkyl group to about 6 carbon atoms.In certain embodiments, the intramolecularly connection can be disulfide linkage, amido linkage, imine linkage, amine key, alkyl bond and thiazolinyl key.In certain embodiments, X and Y are independently selected from Ser, Asp, Glu, Lys, Om or Cys.In certain embodiments, X and Y are Cys and Cys.In other embodiments, X and Y are Ser and Ser.In other embodiments, X and Y are Asp and Lys or Lys and Asp.

The Xaa1 sequence comprises 3,4,5 or 6 amino acid whose aminoacid sequences between X and the Y.In certain embodiments, the Xaa1 sequence comprises and is being close to the aminoacid sequence that Y has one or more replacements or do not replace the zone of hydroxyl residue.For example, hydroxyl residue district can have at least 2 in 3 contiguous amino acid of Y, itself or be Ser, perhaps be Thr.Other amino acid in the Xaa1 sequence can be arbitrary amino acid.In certain embodiments, the Xaa1 sequence is 3 amino acid.In other embodiments, the Xaa1 sequence is 4 amino acid.In other embodiments, the Xaa1 sequence is 5 amino acid.In other embodiments, the Xaa1 sequence is 6 amino acid.Therefore, Xaa1 can be expressed as Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7 (SEQ ID NO:5).In certain embodiments, Xaa2, Xaa3 and/or Xaa4 can not exist.In certain embodiments, Xaa5, Xaa6 and Xaa7 comprise the residue district of hydroxyl.Therefore, at least two in 3 amino acid can be Ser, hSer, Thr, other Thr, d-Thr or other its non-natural analogue.Xaa2 can be arbitrary amino acid or does not exist, and Xaa3 can be arbitrary amino acid or do not exist, and Xaa4 can be arbitrary amino acid or do not exist; If Xaa6 is that Ser or Thr and Xaa7 are Ser or Thr, then Xaa5 can be arbitrary amino acid; If Xaa5 is that Ser or Thr and Xaa7 are Ser or Thr, then Xaa6 can be arbitrary amino acid; If Xaa5 is that Ser or Tbr and Xaa6 are Ser or Thr, then Xaa7 can be arbitrary amino acid.Therefore, in certain embodiments, Xaa1 can be expressed as that Xaa2 does not exist, Xaa3 is Ala, Gly, Ser, Asp or do not exist, and Xaa4 is Asn, Ala, Asp, Gly or does not exist; Xaa5 is Ala, Leu, Thr or Ser; Xaa6 is Ala, Ser or Thr; And Xaa7 be Ala, Ser, Val, Hse, (S)-2-amino-3-hydroxyl-methylbutyric (Ahb), (2S, 3R)-2-amino-3 hydroxyls-methylvaleric acid (Ahp), D-Thr, Thr or derivatives thereof.In other embodiments, Xaa1 can be expressed as Xaa2 and not exist, and Xaa3 is Ser, Gly or does not exist that Xaa4 is Asn or Asp, and Xaa5 is Ala, Ser, Thr or Leu, and Xaa6 is Ala, Thr or Ser, and Xaa7 is Ser, D-Thr, other Thr or Thr.In certain embodiments, ring district comprise wherein Xaa3 be Ala, wherein Xaa3 be Ser or wherein Xaa3 be the above-mentioned Xaa1 expression formula of Gly.Perhaps or in addition, the ring district comprise wherein Xaa4 be Ala, wherein Xaa4 be Asn, wherein Xaa4 be Asp or wherein Xaa4 be the above-mentioned Xaa1 expression formula of Gly.Perhaps or in addition, the ring district comprise wherein Xaa5 be Ala, wherein Xaa5 be Thr or wherein Xaa5 be the above-mentioned Xaa1 expression formula of Leu.Perhaps or in addition, the ring district comprise wherein Xaa6 be Ser or wherein Xaa6 be the above-mentioned Xaa1 expression formula of Ala.Perhaps or in addition, the ring district comprise wherein Xaa7 be Thr or wherein Xaa7 be the above-mentioned Xaa1 expression formula of D-Thr.Further imagination structure in the ring district is no more than 1,2 or 3 modification, for example replaces, inserts, disappearance and/or derivation.

It should be noted that in whole application, all with the record of Ma Kushi group, for example, each amino acid position all contains the possible amino acid more than 1 in alternative selection.Specifically, each member in the imagination Ma Kushi group should be considered separately, constitutes another embodiment of the invention thus, can not be used as single unit to the Ma Kushi group.

The example in ring of the present invention district includes but not limited to C-N-T-A-T-C (SEQ ID NO:6), C-A-T-A-T-C (SEQ ID NO:7), C-D-T-A-T-C (SEQ ID NO:8), C-G-T-A-T-C (SEQ ID NO:9), C-N-A-A-T-C (SEQ ID NO:10), C-N-T-S-T-C (SEQ ID NO:11), C-N-T-A-dThr-C (SEQ ID NO:12), C-N-T-A-T (OPO3H2)-C (SEQ ID NO:13), C-N-T-A-S-C (SEQ ID NO:14), C-N-T-A-A-C (SEQ ID NO:15), C-N-T-A-V-C (SEQ ID NO:16), C-N-T-A-Hse-C (SEQ ID NO:17), C-N-T-A-Ahb-C (SEQ ID NO:18), C-N-T-A-Ahp-C (SEQ ID NO:19), C-S-N-L-S-T-C (SEQ ID NO:20), C-G-N-L-S-T-C (SEQ ID NO:21), C-A-N-L-S-T-C (SEQ ID NO:22), C-S-A-L-S-T-C (SEQ ID NO:23), C-S-N-A-S-T-C (SEQ ID NO:24), C-S-N-L-A-T-C (SEQ ID NO:25) and C-S-N-L-S-A-C (SEQ ID NO:26).As previously pointed out, further imagination structure in the ring district is no more than 1,2 or 3 modification, for example replaces, inserts, disappearance and/or derivation.

Modification or other amino acid can further be contained at the N-end in the ring district of novel cpd.Such modification comprises the interpolation compound; for example Lys, Ala, Phe, Ile, Ser, octyl group glycine, dissident's acyl group (Isocap), Fmoc-3; 6-dioxy base is sad, Fmoc-1-is amino-4; 7,10-trioxa-13-tridecyl amine succinamic acid, ethanoyl and/or be used for the group of solvability, transmission, signal transduction.Representational trimming loop comprises to Xaa1 sequence interpolation Lys or to Xaa1 sequence interpolation Ile.For example, the ring district of modification can be K-C-N-T-A-T-C (SEQID NO:27).In certain embodiments, add and/or modify in the terminal acid of ring district N-and can change the ring district.For example; ring of the present invention district can followingly modify: ring (2; 7) 1-7 hAmylin, ring (2Asp7Lys) 1-7 hAmylin, N-dissident's acyl group 1-7 hAmylin, N-3; 6 two oxa-capryloyl 1-7 hAmylin, L-octyl group glycine 1-7 hAmylin, wherein Agy is the ethanoyl (2Agy of allylglycine; 7Agy) 1-7 hAmylin, ethanoyl (1Ala) 1-7 hAmylin, (1Thr3Asp) 1-7 hAmylin, dissident's acyl group (7Ala) 5-7 sCT, ethanoyl (2Agy; 7Agy) 1-7 sCT and ring (1,7) (1Asp 7Lys) 1-7 sCT.Therefore, be example with dissident's acyl group (7Ala) 5-7 sCT, certain embodiments of the present invention comprise modification at the N end regions in ring of the present invention district, make Xaa2-Xaa5 not exist.

In whole application, described aminoacid sequence can be called the amino acid of a position of contiguous reference peptide to b position.In this application, the reference peptide is islet amyloid polypeptide and thyrocalcitonin, and its sequence is shown in SEQ ID NO:1-4.For example, in the paragraph formerly, 1-7 hAmylin refers to comprise the aminoacid sequence (SEQ ID NO:1) of 1 to 7 of people's islet amyloid polypeptide.Modification to the reference peptide can followingly be explained: in abutting connection with the modifier bit of modifying.For example, (2Asp 7Lys) 1-7hAmylin represents the aminoacid sequence of people's islet amyloid polypeptide 1-7 position, has the modification that is become Asp by Cys at 2, has the modification that is become Lys by Cys at 7.

The alpha helical region of novel cpd can be about 8-23 amino acid long.In certain embodiments, alpha helical region is amphiphatic.In certain embodiments, alpha helical region contains about 3-6 spiral corner.In certain embodiments, alpha helical region contains 3,4,5 or 6 spiral corners.In other embodiments, alpha helical region is and about 3,4,5 or 6 rigid structures that the spiral corner is equal to.The example of ideal spiral is LLQQLQKLLQKLKQY (SEQ IDNO:28).In certain embodiments, the α spiral is amphiphilic structure.Therefore, can select to provide the amino acid needed feature of this type structure.

Have found that, in the alpha helical region of novel cpd, need combination or its part and/or some CGRP element of thyrocalcitonin alpha helical region, islet amyloid polypeptide and thyrocalcitonin alpha helical region.As the situation in ring district, the imagination alpha helical region can be from any islet amyloid polypeptide or thyrocalcitonin and analogue thereof.Therefore, in certain embodiments, alpha helical region is at least a portion of thyrocalcitonin or calcitonin-like alpha helical region.In other embodiments, alpha helical region is at least a portion of thyrocalcitonin or calcitonin-like alpha helical region and at least a portion of islet amyloid polypeptide or islet amyloid polypeptide analogue alpha helical region.In other embodiments, the alpha helical region of novel cpd comprises the element of CGRP.Further the imagination novel cpd can have and is no more than 1,2,3,4,5,6,7,8,9 or 10 and further modifies, and for example replaces, inserts, disappearance and/or derivation.

In certain embodiments, alpha helical region of the present invention can comprise 8 amino acid of 18,19,20,21,22,23,24,25,26 or 27 to sCT of sCT.And alpha helical region can comprise more than 1 part of the thyrocalcitonin or the calcitonin-like alpha helical region of identical or different species, for example 8-21 sCT 19-27 sCT; 8-21 sCT 18-27 sCT; Or 8-16 hCT 17-27 sCT; Or (11Arg) 8-16 hCT (18Arg) 17-27 sCT.Perhaps or in addition, the α spiral of above-mentioned 8-18 sCT to 8-27 sCT can further comprise one or more displacements or its arbitrary combination among (10Aib), (11Arg), (11Orn), (11hArg), (11Cit), (11hLys), (11Lys (for)), (17Aib), (18Arg), (18Orn), (18hArg), (18Cit), (18hLys), (18Lys (for)), (18Lys (PEG5000)), (22Leu), (24Pro).

In one embodiment, alpha helical region of the present invention can be expressed as (alpha helical region I type) R1-VL Xaa10 Xaa11 LSQ Xaa15 L Xaa17 Xaa18 LQT Xaa22 P Xaa24TNT-R1 (SEQ ID NO:29), wherein

Xaa10 is Gly or Aib;

Xaa11 is Lys, Arg, Orn, hArg, Cit, hLys or Lys (for);

Xaa15 is Glu or Phe;

Xaa17 is His or Aib;

Xaa18 is Lys, Arg, Orn, hArg, Cit, hLys, Lys (for), Lys (PEG5000);

Xaa22 is Try or Leu;

Xaa24 is Arg or Pro; Or

There is not or comprises 1-4 additional amino acid in R1.

In addition, what should remember is that each member or its combination in the Ma Kushi group all are another embodiment of the invention, can not be understood that single unit.As an example, a shorthand that is used to illustrate is arranged, embodiment of the present invention comprise alpha helical region I pattern, and wherein Xaa18 can be Lys, Arg, Orn, hArg, Cit, hLys or Lys (for), and each variant all is an independent embodiment of the present invention.Therefore, alpha helical region I pattern has an embodiment, and wherein Xaa18 is Lys.Another embodiment is arranged, and wherein Xaa18 is Arg or the like.Further the imagination alpha helical region can comprise and is no more than 1,2,3,4,5,6,7,8,9 or 10 modification, for example replaces, inserts, disappearance and/or derivation.Therefore, alpha helical region I type compound can further have disappearance at C-terminal.In certain embodiments, the amino acid of R1 can form α spiral corner.

The example of alpha helical region I type of the present invention includes but not limited to 8-18 sCT, 8-21 sCT, 8-24sCT, 8-27 sCT, (11Arg) 8-18 sCT, (18Arg) 8-18 sCT, (11Arg 18Arg) 8-18sCT, (11Orn 18Orn) 8-18 sCT, (11Arg 18Cit) 8-18 sCT, (11hArg 18hArg) 8-18 sCT, (11Arg 18Orn) 8-18 sCT, (11Cit 18Arg) 8-18 sCT, (11Cit18Cit) 8-18 sCT, (11hLys 18hLys) 8-18 sCT, (10Aib 11Arg 17Aib 18Arg) 8-18 sCT, (11Lys (for) 18Lys (for)) 8-18 sCT, (10Aib 11Lys (for) 17Aib18Lys (for)) 8-18 sCT, (11Arg 18Lys (PEG 5000)) 8-18 sCT, (11Arg) 8-21 sCT, (18Arg) 8-21 sCT, (11Arg 18Arg) 8-21 sCT, (11Orn 18Orn) 8-21sCT, (11Arg 18Cit) 8-21 sCT, (11hArg 18hArg) 8-21 sCT, (11Arg 18Orn) 8-21 sCT, (11Cit 18Arg) 8-21 sCT, (11Cit 18Cit) 8-21 sCT, (11hLys18hLys) 8-21 sCT, (10Aib 11Arg 17Aib 18Arg) 8-21 sCT, (11Lys (for) 18Lys (for)) 8-21 sCT, (10Aib 11Lys (for) 17Aib 18Lys (for)) 8-21 sCT, (11Arg 18Lys (PEG 5000)) 8-21 sCT, (11Arg) 8-24 sCT, (18Arg) 8-24sCT, (11Arg 18Arg) 8-24 sCT, (11Arg 18Arg 22Leu) 8-24 sCT, (11Arg18Arg 24Pro) 8-24 sCT, (11Orn 18Orn) 8-24 sCT, (11Arg 18Cit) 8-24sCT, (11hArg 18hArg) 8-24 sCT, (11Arg 18Orn) 8-24 sCT, (11Cit 18Arg) 8-24 sCT, (11Cit 18Cit) 8-24 sCT, (11hLys 18hLys) 8-24 sCT, (10Aib11Arg 17Aib 18Arg) 8-24 sCT, (11Lys (for) 18Lys (for)) 8-24 sCT, (10Aib11Lys (for) 17Aib 18Lys (for)) 8-24 sCT, (11Arg 18Lys (PEG 5000)) 8-24sCT, (11Arg) 8-27 sCT, (18Arg) 8-27 sCT, (11Arg 18Arg) 8-27 sCT, (11Arg 18Arg 22Leu) 8-27 sCT, (11Arg 18Arg 24Pro) 8-27 sCT, (11Orn18Orn) 8-27 sCT, (11Arg 18Cit) 8-27 sCT, (11hArg 18hArg) 8-27 sCT, (11Arg 18Orn) 8-27 sCT, (11Cit 18Arg) 8-27 sCT, (11Cit 18Cit) 8-27sCT, (11hLys 18hLys) 8-27 sCT, (10Aib 11Arg 17Aib 18Arg) 8-27sCT, (11Lys (for) 18Lys (for)) 8-27 sCT, (10Aib 11Lys (for) 17Aib18Lys (for)) 8-27 sCT, (11Arg 18Lys (PEG 5000)) 8-27 sCT, (11Arg18Arg) 8-21 sCT-19-27 sCT and (11Arg 18Arg) 8-21 sCT-(18Leu) 18-27sCT.

In certain embodiments, alpha helical region of the present invention can comprise the part of islet amyloid polypeptide or islet amyloid polypeptide analogue alpha helical region and the part of thyrocalcitonin or calcitonin-like alpha helical region.Alpha helical region of the present invention can comprise 8 of hAmylin 18,19,20,21,22,23,24,25,26 or 27 amino acid to 13,14,15,16,17,18 and 19 the amino acid of 11,12,13,14,15,16,17,18 or 19 the amino acid of hAmylin and sCT to sCT.Perhaps or in addition, the alpha helical region of above-mentioned islet amyloid polypeptide and thyrocalcitonin can further comprise (8Val), (9Leu), (9Met), (10Gly), (10His), (12Thr), (13Thr), (13Asn), (13Phe), (13Tyr), (14Arg), (14Ala), (14Asp), (14Glu), (14Gln), (14Thr), (14Gly), (15Leu), (15Ser), (15Glu), (15Ala), (15Tyr), (16Asp), (17Ser), (17Phe), (18Arg), (17Aib), (18Arg), (18Orn), (18hArg), (18Cit), (18hLys), (18Lys (for)), (18Lys (PEG5000)), (19Phe), (20His), (21Asn), (22Met), (22Val), (22Phe), (22Leu), one or more displacements (24Pro) or its arbitrary combination.In certain embodiments, the amino acid number in the alpha helical region of the present invention is at least 10 amino acid.In other embodiments, the amino acid number in the alpha helical region of the present invention is 11,12,13,14,15,16,17,18,19,20,21,22 or 23.In other embodiments, the amino acid number in the alpha helical region of the present invention is 24 or more.

In one embodiment, alpha helical region of the present invention can be expressed as (alpha helical region II type) R1-Xaa8 Xaa9 Xaa10 R Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18Xaa19 Xaa20 Xaa21 Xaa22 P Xaa24 TNT-R1 (SEQ ID NO:30), wherein

Xaa8 is Ala or Val;

Xaa9 is Thr, Met or Leu;

Xaa10 is Gln, Gly, His;

Xaa12 is Leu or Thr;

Xaa13 is Ala, Thr, Asn, Phe, Tyr, Ser or Thr;

Xaa14 is Asn, Arg, Ala, Asp, Glu, Gln, Thr or Gly;

Xaa15 is Phe, Leu, Ser, Glu, Ala, Asp or Tyr;

Xaa16 is Leu or Asp;

Xaa17 is Val, His, Ser, Phe or Aib;

Xaa18 is His, Arg, Lys, Orn, hArg, Cit, hLys, Lys (for) or Lys (PEG5000);

Xaa19 is Leu, Ser or Phe;

Xaa20 is Gln or His;

Xaa21 is Thr or Asn;

Xaa22 is Tyr, Val, Phe, Leu or Met;

Xaa24 is Arg or Pro; And

R1 is not for existing or comprise 1-4 additional amino acid.

In addition, what should remember is that each member or its combination in the Ma Kushi group all are another embodiment of the invention, can not be understood that single unit.Further the imagination alpha helical region can comprise this paper and describes 1,2,3,4,5,6,7,8,9 or 10 modification that is no more than in the compound, and for example replaces, inserts, disappearance and/or derivation.For example, in certain embodiments, alpha helical region II type compound can have disappearance at C-terminal, causes 27,26,25,24 or 22 disappearances.But in other embodiments, disappearance is not removed 19,20,21 or 22 amino acid.

The example of II type alpha helical region includes but not limited to (8Val 9Leu 10Gly) 11-15hAmylin 16-27 sCT, (8Val 9Leu 10GLy) 11-15 hAmylin (18Arg) 16-27sCT, 8-12 hAmylin (18Arg) 13-27 sCT, 8-18 hAmylin 19-23 sCT, 8-18HAmylin 19-27 sCT, (15Glu 18Arg) 8-18 hAmylin 19-24 sCT, (14Arg15Ser) 8-18 hAmylin 19-22 sCT, (13Ala 14Ala 15Ala) 8-18 hAmylin19-27 sCT, (13Ala 14Asp 15Ala) 8-18 hAmylin 19-22 sCT, (13Ala 14Asp) 8-18 hAmylin 19-23 sCT, (13Ala 14Asp) 8-18 hAmylin 19-27 sCT, (13Ala 14Ala) 8-18 hAmylin 19-22 sCT, (13Ala 14Glu) 8-18 hAmylin19-22 sCT, (13Thr 14Asp 15Tyr) 8-18 hAmylin 19-22 sCT, (13Ala 14Gln) 8-18 hAmylin 19-22 sCT, (13Asn 14GLu 15Tyr) 8-18 hAmylin 19-27sCT, (13Phe 14Asp) 8-18 hAmylin 19-27 sCT, (13Ala 14Asp) 8-18hAmylin (15Glu 18Arg) 8-18 hAmylin 19-24 sCT, (19Phe 22Phe) 19-27sCT, (13Ala 14Asp) 8-18 hAmylin (19Phe 20His 22Phe) 19-27 sCT, (13Ala 14Asp) 8-18 hAmylin (19Phe 22Phe) 19-27 sCT, (9Thr 10His) 8-18 hAmylin 19-22 sCT, (9Thr 10His 14Gly 15Leu 17Ser 18Arg) 8-19hAmylin 20-23 sCT, 8-18 hAmylin (21Asn 22Phe 23Val) 19-23 sCT, 8-18 hAmylin (22Met) 19-27 sCT, 8-18 hAmylin (22Val) 19-27 sCT, (9Met 12Thr 13Tyr 14Thr 15Glu 16Asp 17Phe) 8-17 hAmylin (18Arg) 18-20 sCT.In other embodiments, novel cpd comprises the variant of above-mentioned representative compounds, and it has at 22,23,24,25,26 or 27 bit position terminated α spirals corresponding to sCT.In other words, also specifically described compound 8-18 hAmylin 19-24sCT, because this compound only is to be truncated to 24 above-mentioned 8-18 hAmylin 19-27sCT.As another example, also specifically described compound (13Ala 14Asp 15Ala) 8-18hAmylin 19-23, because above statement is applicable to (13Ala 14Asp 15Ala) 8-18hAmylin 19-22.

In certain embodiments, C end tail of the present invention comprises 27,28,29,30,31,32 or 33 to 36 or 37 the amino acid of hAmylin.In other embodiments, C end tail comprises 27 or 28 to 32 the amino acid of sCT; But during from thyrocalcitonin or calcitonin-like, last position of C end tail is not Pro, Hyp, homoserine (Hse) or Hse derivative from thyrocalcitonin or calcitonin-like, alpha helical region in the Dang Huan district.Perhaps or in addition, above-mentioned islet amyloid polypeptide and thyrocalcitonin alpha helical region can further comprise one or more replacements or its arbitrary combination among (27Tyr) hAmylin, (29Arg) hAmylin, (32Val) hAmylin, (32Thr) hAmylin, (34Glu) hAmylin, (35Lys) hAmylin, (36Phe) hAmylin, (36Ala) hAmylin, (37Phe) hAmylin, (30Asn) sCT, (32Tyr) sCT.

In one embodiment, C end tail of the present invention can be expressed as Xaa28 Xaa29Xaa30 Xaa31 Xaa32 Xaa33 G Xaa35 Xaa36 Xaa37 Xaa38 (SEQ IDNO:31), wherein

Xaa28 is Lys, Tyr or does not exist;

Xaa29 is Ser, Pro or does not exist;

Xaa30 is Ser, Pro, Arg or does not exist;

Xaa31 is Thr or does not exist;

Xaa32 is Asn or does not exist;

Xaa33 is Val, Thr or does not exist;

Xaa35 is Ser, Glu;

Xaa36 is Asn, Lys or Gly;

Xaa37 is Thr, Phe or Ala;

Xaa38 is Tyr, Phe, Pro or does not exist;

When precondition was the Dang Huan district from thyrocalcitonin or calcitonin-like, alpha helical region from thyrocalcitonin or calcitonin-like, last position of C end tail was not Pro, Hyp, homoserine (Hse) or Hse derivative.

In addition, what should remember is that each member or its combination in the Ma Kushi group all are another embodiment of the invention, can not be understood that single unit.Further imagination C end tail can comprise 1,2 or 3 modification that is no more than in the described compound of first previous paragraphs, for example replaces, inserts, disappearance and/or derivation.

The example of C end tail of the present invention includes but not limited to 27-37 rAmylin, (27Tyr 29Arg32Thr) 27-37 rAmylin, (29Arg 32Thr) 28-37 rAmylin, 30-37 hAmylin, (32Thr) 30-37 hAmylin, (35Lys 36Ala 37Phe) 30-37 hAmylin, 30-36hAmylin, (32Val) 30-36 hAmylin, (34Glu 36Phe) 30=36 hAmylin, 31-37 hAmyin, 31-36 hAmylin, 33-36 hAmylin, 33-7 hAmylin, 28-32 sCT, (30Asn 32Tyr) 28-32 sCT and 27-32 sCT.In other embodiments, C end tail comprises aminoacid sequence KSNFVPTN (SEQ ID NO:32) or SNFVPTNV (SEQ IDNO:33).

Can be as described in the first previous paragraphs, further imagination is no more than 1,2 or 3 modification to C end tail structure of the present invention, for example replaces, inserts, disappearance and/or derivation.The C end tail of novel cpd can further comprise at C-terminal to be modified or additional amino acid.Such modification comprises the interpolation compound, for example Lys (until 4 Lys), L-octyl group glycine, 4ABU (4-aminobutyric acid), 9Anc (9 aminononanoic acid) and/or be used for the group of solvability, stability or transmission.Example comprises 33-37 hAmylin L-octyl group glycine, 33-37 hAmylin 4ABU and 33-37 hAmylin 9Anc.

Aspect general, compound of the present invention comprises

(a) any ring district of the present invention;

(b) any alpha helical region of the present invention; With

When (c) any C end tail of the present invention, precondition were Dang Huan districts from thyrocalcitonin or calcitonin-like, alpha helical region from thyrocalcitonin or calcitonin-like, last of C end tail was not Pro, Hyp, homoserine (Hse) or Hse derivative.

Another general aspect, compound of the present invention comprises

(a) comprise Xaa1 or have the ring district of the Xaa1 of modification at the N-end;

(b) comprise the alpha helical region of I type or II type alpha helical region;

(c) the C end tail of representing by SEQ ID NO:31, when precondition was the Dang Huan district from thyrocalcitonin or calcitonin-like, alpha helical region from thyrocalcitonin or calcitonin-like, last position of C end tail was not Pro, Hyp, homoserine (Hse) or Hse derivative.C-terminal can comprise further modification.

More on the one hand, compound of the present invention comprises the aminoacid sequence of formula I: Xaa1 XXaa3 Xaa4 Xaa5 Xaa6 Y Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ IDNO:34), wherein

Xaa1 is A, C, hC, D, E, F, I, L, K, hK, R, hR, S, Hse (homoserine), T, G, Q, N, M, Y, W, P, Hyp (oxyproline), H, V or does not exist;

Xaa3 is A, D, E, N, Q, G, V, R, K, hK, hR, H, I, L, M or does not exist;

Xaa4 is A, I, L, S, Hse, T, V, M or does not exist;

Xaa5 is A, S, T, Hse, Y, V, I, L or M;

Xaa6 is T, A, S, Hse, Y, V, I, L or M;

Xaa8 is A, V, I, L, F or M;

Xaa9 is L, T, S, Hse, V, I or M;

Xaa10 is G, H, Q, K, R, N, hK or hR;

Xaa11 is K, R, Q, N, hK, hR or H;

Xaa12 is L, I, V, F, M, W or Y;

Xaa13 is A, F, Y, N, Q, S, Hse or T;

Xaa14 is A, D, E, G, N, K, Q, R, H, hR or hK;

Xaa15 is A, D, E, F, L, S, Y, I, V or M;

Xaa16 is L, F, M, V, Y or I;

Xaa17 is H, Q, N, S, Hse, T or V;

Xaa18 is K, hK, R, hR, H, u (Cit) or n (Orn);

Xaa19 is F, L, S, Hse, V, I, T or does not exist;

Xaa20 is H, R, K, hR, hK, N, Q or does not exist;

Xaa21 is T, S, Hse, V, I, L, Q, N or does not exist;

Xaa22 is F, L, M, V, Y or I;

Xaa23 is P or Hyp;

Xaa24 is P, Hyp, R, K, hR, hK or H;

Xaa25 is T, S, Hse, V, I, L, F or Y;

Xaa26 is N, Q, D or E;

Xaa27 is T, V, S, F, I or L;

Xaa28 is G or A;

Xaa29 is S, Hse, T, V, I, L or Y;

Xaa30 is E, G, K, N, D, R, hR, hK, H or Q;

Xaa31 is A, T, S, Hse, V, I, L, F or Y; With

Xaa32 is F, P, Y, Hse, S, T or Hyp;

Wherein X and Y can produce key, and are the independent residue of selecting with following side chain: thus the chemical bonding formation intramolecularly connection each other of described side chain, for example disulfide linkage; Amido linkage; Can form the alkyl acid and the alkylamine of cyclic lactames; Alkyl aldehydes or alkylogen and alkylamine, but its condensation and being reduced, thus alkylamine or imine linkage formed; Or thereby described side chain can connect formation alkyl, thiazolinyl, alkynyl, ether or thioether bond.Alkyl chain can comprise having about 1 low alkyl group to about 6 carbon atoms.In certain embodiments, the intramolecularly connection can be disulfide linkage, amido linkage, imine linkage, amine key, alkyl bond and ethylene linkage.In certain embodiments, X and Y are independently selected from Ser, Asp, Glu, Lys, Orn or Cys.In certain embodiments, X and Y are Cys and Cys.In other embodiments, X and Y are Ser and Ser.In other embodiments, X and Y are Asp and Lys or Lys and Asp.

More on the one hand, compound of the present invention comprises the aminoacid sequence of formula II: Xaa1 Xaa2Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ IDNO:35), wherein

Xaa1 is A, C, D, F, I, K, S, T or does not exist;

Xaa2 is C, D, S or does not exist;

Xaa3 is A, D, N or does not exist;

Xaa4 is A, L, T or does not exist;

Xaa5 is A or S;

Xaa6 is T, A, S or V;

Xaa7 is C, K or A;

Xaa8 is A, V, L or M;

Xaa9 is L or T;

Xaa10 is G, H or Q;

Xaa11 is K, R, Q or hArg;

Xaa12 is L, W or Y;

Xaa13 is A, F, N, Q, S or T;

Xaa14 is A, D, E, G, N, K, Q or R;

Xaa15 is A, D, E, F, L, S or Y;

Xaa16 is L or F;

Xaa17 is H, Q, S or V;

Xaa18 is K, R, hArg, u (Cit) or n (Orn);

Xaa19 is F, L, S or does not exist;

Xaa20 is H, Q or does not exist;

Xaa21 is T, N or does not exist;

Xaa22 is F, L, M, V or Y;

Xaa23 is P;

Xaa24 is P or R;

Xaa25 is T;

Xaa26 is N;

Xaa27 is T or V;

Xaa28 is G;

Xaa29 is S;

Xaa30 is E, G, K or N;

Xaa31 is A or T; With

Xaa32 is F, P or Y.

More on the one hand, compound of the present invention comprises the aminoacid sequence of formula III: Xaa1 Xaa2Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ IDNO:36), wherein

Xaa1 is A, C, F, I, K, S or does not exist;

Xaa2 is C, D or S;

Xaa3 is A, D or N;

Xaa4 is A, L or T;

Xaa5 is A or S;

Xaa6 is T;

Xaa7 is C or K;

Xaa8 is A or V;

Xaa9 is L or T;

Xaa10 is G, H or Q;

Xaa11 is K, R or hArg;

Xaa12 is L;

Xaa13 is A, F, N, S or T;

Xaa14 is A, D, E, G, N, Q or R;

Xaa15 is A, E, F, L, S or Y;

Xaa16 is L;

Xaa17 is H, S or V;

Xaa18 is K, R, hArg, u (Cit) or n (Orn);

Xaa19 is F, L or S;

Xaa20 is H or Q;

Xaa21 is T or N;

Xaa22 is F, L, M, V or Y;

Xaa23 is P;

Xaa24 is P or R;

Xaa25 is T;

Xaa26 is N;

Xaa27 is T or V;

Xaa28 is G;

Xaa29 is S;

Xaa30 is E, G, K or N;

Xaa31 is A or T; With

Xaa32 is F, P or Y.

Aspect general, the sequence of formula I, II or III further comprise 1,2,3,4,5,6,7,8,9,10,11,12 or more a plurality of displacement, insertion, extension and/or derivation modify.In certain embodiments, the sequence of formula I, II or III is included in the Val that inserts between 22 and 23 amino acids.In other embodiments, the sequence of formula I, II or III is included in the Gln that inserts between 22 and 23.In other embodiments, the sequence of formula I, II or III is included in Gln-Thr-Tyr (the SEQ ID NO:37) sequence between 22 and 23.In other embodiments, the sequence of formula I, II or III is included in Leu-Gln-Thr-Tyr (the SEQ ID NO:38) sequence between 22 and 23.Another general aspect, the modification of formula I, II or III can be in the N-end.In certain embodiments, the N end parts of formula I, II or III has the octyl group glycine of interpolation.In other embodiments, the N end parts of formula I, II or III has dissident's acyl group of interpolation.

More on the one hand, compound of the present invention comprises the aminoacid sequence of formula IV: Xaa1 Xaa2Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ IDNO:39), wherein

Xaa1 is A, C, D, F, K, T or does not exist;

Xaa2 is A, C, D, S or does not exist;

Xaa3 is A, D, N or does not exist;

Xaa4 is A, L, T or does not exist;

Xaa5 is A or S;

Xaa6 is A, S, T or V;

Xaa7 is A, C or K;

Xaa8 is A, L, M or V;

Xaa9 is L or T;

Xaa10 is G, H or Q;

Xaa11 is K, Q or R;

Xaa12 is L, W or Y;

Xaa13 is A, N, Q, S or T;

Xaa14 is A, D, E, G, K, N, Q or R;

Xaa15 is A, D, E, F, L, S or Y;

Xaa16 is F or L;

Xaa17 is H, Q, S or V;

Xaa18 is K or R;

Xaa19 is F, L, S or does not exist;

Xaa20 is H, K, Q or does not exist;

Xaa21 is Q, T or does not exist;

Xaa22 is F, L or Y;

Xaa23 is P;

Xaa24 is P or R;

Xaa25 is T;

Xaa26 is N;

Xaa27 is T or V;

Xaa28 is G;

Xaa29 is S;

Xaa30 is E, K or N;

Xaa31 is A or T;

Xaa32 is F, Y or does not exist;

Aspect general, the sequence of formula IV further comprise 1,2,3,4,5,6,7,8,9,10,11,12 or more a plurality of displacement, insertion, extension and/or derivation modify.In certain embodiments, the sequence of formula I, II, III or IV is included in 24 disappearance.

More on the one hand, compound of the present invention comprises aminoacid sequence, and it contains

(a) comprise the ring district of Xaa1;

(b) α spiral ring I type; With

(c) C end tail;

Wherein X1 comprises aminoacid sequence X Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Y (SEQ ID NO:5), wherein,

Xaa2 is arbitrary amino acid or does not exist;

Xaa3 is Ala, Gly, Ser, Asp or does not exist;

Xaa4 is Asn, Ala, Asp, Gly or does not exist;

Xaa5 is Ala, Leu, Thr or Ser;

Xaa6 is Ala, Ser or Thr; With

Xaa7 be Ala, Ser, Val, Hse, (S)-2-amino-3-hydroxyl-methylbutyric (Ahb), (2S, 3R)-2-amino-3 hydroxyls-methylvaleric acid (Ahp), D-Thr, Thr or derivatives thereof;

X and Y are the amino acid that can produce key, are the independent residue of selecting with following side chain: thus the chemical bonding formation intramolecularly connection each other of described side chain, for example disulfide linkage; Amido linkage; Can form the alkyl acid and the alkylamine of cyclic lactames; Alkyl aldehydes or alkylogen and alkylamine, but its condensation and being reduced, thus alkylamine or imine linkage formed; Or thereby described side chain can connect formation alkyl, thiazolinyl, alkynyl, ether or thioether bond;

Alpha helical region I type comprises sequence R1-V L Xaa10 Xaa11 L S Q Xaa15 L Xaa17Xaa18 L Q T Xaa22 P Xaa24 T N T-R1 (SEQ ID NO:29), wherein

Xaa10 is Gly or Alb;

Xaa11 is Lys, Arg, Orn, hArg, Cit, hLys or Lys (for);

Xaa15 is Glu or Phe;

Xaa17 is His or Aib;

Xaa18 is Lys, Arg, Orn, hArg, Cit, hLys, Lys (for), Lys (PEG5000);

Xaa22 is Try or Leu;

Xaa24 is Arg or Pro; Or

There is not or comprises 1-4 additional amino acid in R1; With

C end tail comprises sequence X aa28 Xaa29 Xaa30 Xaa31 Xaa32 Xaa33 G Xaa35Xaa36 Xaa37 Xaa38 (SEQ ID NO:31), wherein

Xaa28 is Lys, Tyr or does not exist;

Xaa29 is Ser, Pro or does not exist;

Xaa30 is Ser, Pro, Arg or does not exist;

Xaa31 is Thr or does not exist;

Xaa32 is Asn or does not exist;

Xaa33 is Val, Thr or does not exist;

Xaa35 is Ser, Glu;

Xaa36 is Asn, Lys or Gly;

Xaa37 is Thr, Phe or Ala;

Xaa38 is Tyr Phe, Pro or does not exist;

When precondition was the Dang Huan district from thyrocalcitonin or calcitonin-like, alpha helical region from thyrocalcitonin or calcitonin-like, last position of C end tail was not Pro, Hyp, homoserine (Hse) or Hse derivative.

More on the one hand, compound of the present invention comprises aminoacid sequence, and it contains

(a) comprise the ring district of Xaa1;

(b) α spiral ring II type; With

(c) C end tail;

Its Central District Xaa1 comprises aminoacid sequence X Xaa2 Xaa3 Xaa4 Xaa5 Xaa6Xaa7 Y, wherein

Xaa2 is arbitrary amino acid or does not exist;

Xaa3 is Ala, Gly, Ser, Asp or does not exist;

Xaa4 is Asn, Ala, Asp, Gly or does not exist;

Xaa5 is Ala, Leu, Thr or Ser;

Xaa6 is Ala, Ser or Thr; With

Xaa7 be Ala, Ser, Val, Hse, (S)-2-amino-3-hydroxyl-methylbutyric (Ahb), (2S, 3R)-2-amino-3 hydroxyls-methylvaleric acid (Ahp), D-Thr, Thr or derivatives thereof;

X and Y are the amino acid that can produce key, are the independent residue of selecting with following side chain: thus the chemical bonding formation intramolecularly connection each other of described side chain, for example disulfide linkage; Amido linkage; Can form the alkyl acid and the alkylamine of cyclic lactames; Alkyl aldehydes or alkylogen and alkylamine, but its condensation and being reduced, thus alkylamine or imine linkage formed; Or thereby described side chain can connect formation alkyl, thiazolinyl, alkynyl, ether or thioether bond;

Alpha helical region II type comprises sequence R1-Xaa8 Xaa9 Xaa10 R Xaa12 Xaa13Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 P Xaa24TNT-R1 (SEQ ID NO:30), wherein

Xaa8 is Ala or Val;

Xaa9 is Thr, Met or Leu;

Xaa10 is Gln, Gly, His;

Xaa12 is Leu or Thr;

Xaa13 is Ala, Thr, Asn, Phe, Tyr, Ser or Thr;

Xaa14 is Asn, Arg, Ala, Asp, Glu, Gln, Thr or Gly;

Xaa15 is Phe, Leu, Ser, Glu, Ala, Asp or Tyr;

Xaa16 is Leu or Asp;

Xaa17 is Val, His, Ser, Phe or Alb;

Xaa18 is His, Arg, Lys, Orn, hArg, Cit, hLys, Lys (for) or Lys (PEG5000);

Xaa19 is Leu, Ser or Phe;

Xaa20 is Gln or His;

Xaa21 is Thr or Asn;

Xaa22 is Tyr, Val, Phe, Leu or Met;

Xaa24 is Arg or Pro; With

There is not or comprises 1-4 additional amino acid in R1; With

C end tail comprises sequence X aa28 Xaa29 Xaa30 Xaa31 Xaa32 Xaa33 G Xaa35Xaa36 Xaa37 Xaa38 (SEQ ID NO:31), wherein

Xaa28 is Lys, Tyr or does not exist;

Xaa29 is Ser, Pro or does not exist;

Xaa30 is Ser, Pro, Arg or does not exist;

Xaa31 is Thr or does not exist;

Xaa32 is Asn or does not exist;

Xaa33 is Val, Thr or does not exist;

Xaa35 is Ser, Glu;

Xaa36 is Asn, Lys or Gly;

Xaa37 is Thr, Phe or Ala;

Xaa38 is Tyr Phe, Pro or does not exist.

More on the one hand, compound of the present invention comprises:

SEQ ID NO：40 KCNTATCVLGKLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：41 KCNTATCVLGRLSQELHRLQTLPRTNTGSNTY

SEQ ID NO：42 KCNTATCVLGRLSQELHRLQTYPPTNTGSNTY

SEQ ID NO：43 KCNTATCVLGRLSQELHRLQTYPRTNVGSNTY

SEQ ID NO：44 KCNTATCVLGRLSQELHRLQTLPPTNVGSNTY

SEQ ID NO：45 KCNTATCVLGRLANFLHRLQTYPRTNTGSNTY

SEQ ID NO：46 ACNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：47 KCNAATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：48 KCNTAACVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：49 CANLSTCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO:50 dissident acyl group-STAVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：51 CSNASTCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：52 CSNLATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：53 CSNLSACVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：54 KCNTATCVLGRLSQELHKLQTYPRTNTGSNTY

SEQ ID NO：55 KCNTATCVLGRLSQELHRLQTYPRTNTGSGTP

SEQ ID NO：56 CSALSTCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：57 Ac-(Agy)SNLST(Agy)VLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：58 Ac-K(Agy)NTAT(Agy)VLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO:59 dissident acyl group-STAVL (Aib) RLSQELRLQTYPRTNTGSGTP

SEQ ID NO:60 dissident acyl group-STAVLG[K (For)] LSQELH[K (For)] LQTYPRTNTGSGTP

SEQ ID NO:61 dissident acyl group-STAVL (Aib) [K (For)] LSQEL (Aib) [K (For)] LQTYPRTNT GSNTY

SEQ ID NO:62 dissident acyl group-STAVL (Aib) [K (For)] LSQEL (Aib) [K (For)] LQTYPRTNV GSNTY

SEQ ID NO：63 KCNTATCLLQQLQKLLQKLKQYPRTNTGSNTY

SEQ ID NO：64 KCNTASCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：65 KCNTAVCVLGRLSQELHRLQTYPRTnTGSNTY

SEQ ID NO：66 KCNTATCVLGRLSQELHRYPRTNTGSNTY

SEQ ID NO：67 KCNTATCVLGK(For)LSQELHK(For)LQTYPRTNTGSNTY

SEQ ID NO：68 KCNTA(d-Thr)CVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：69 KCNTA(dAh)CVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：70 AC-ACNTATCVLGRLSQELHK(PEG5000)LQTYPRTNTGSNTY

SEQ ID NO：71 KCNTATCVLGRLSQELHRLQTLQTYPRTNTGSNTY

SEQ ID NO：72 KCNTATCVLGRLSQELHRLQTLLQTYPRTNTGSNTY

SEQ ID NO：73 KCNTATCVLGKLSQELHKLQTYPRTNTGSNTY

SEQ ID NO：74 KCNTSTCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：75 KCNTATCATQRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：76 KCNTATCATQRLSQELHRLQTYPRTNVGSNTY

SEQ ID NO：77 KCNTSTCATQRLANELVRLQTYPRTNVGSNTY

SEQ ID NO：78 KCNTA(Hse)CVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：79 KCNTA(Ahb)CVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：80 KCNTA(Ahp)CVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：81 KCNTAT(OPO3H2)CVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：82 KCNTATCVLG(Orn)LSQELH(Orn)LQTYPRTNTGSNTY

SEQ ID NO：83 KCNTATCVLG(Cit)LSQELH(Cit)LQTYPRTNTGSNTY

SEQ ID NO:84 KCNTATCVLG (high K) LSQELH (high K) LQTYPRTNTGSNTY

SEQ ID NO:85 L-octyl group glycine KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO:86 N-[3,6]-two oxa-capryloyl-CNTATCVLGRLSQELHRLQTVPRTNTGSNTY

SEQ ID NO：87 KCNTATCMLGRYTQDFHRLQTYPRTNTGSNTY

SEQ ID NO：88 DSNLSTKVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：89 KDNTATKVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：90 CNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：91 KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(9Anc)

SEQ ID NO:92 KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (L-octyl group glycine)

SEQ ID NO:93 N-dissident acyl group-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO:94 KCNTATCVLG (high R) LSQELH (high R) LQTYPRTNTGSNTY

SEQ ID NO：95 FCNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：96 KCNTATCVLGRLSQELH(Cit)LQTYPRTNTGSNTY

SEQ ID NO：97 KCNTATCVLGRLSQELH(Orn)LQTYPRTNTGSNTY

SEQ ID NO：98 ICNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO:99 1-octyl group glycine-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO:100 dissident acyl group-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：101 KCNTATCVLG(Cit)LSQELHRLQTYPRTNTGSNTY

SEQ ID NO：102 KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(4ABU)

SEQ ID NO:103 dissident acyl group-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (4ABU)

SEQ ID NO：104 KCNTSTCATQRLANELVRLQTYPRTNVGSEAF

SEQ ID NO：105 KCNTATCVLGRLSQELHRLQTYPTNVGSEAF

SEQ ID NO：106 KCNTATCVLGRLSRSLHRLQTYPRTNTGSNTY

SEQ ID NO：107 KCNTATCVTHRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：108 KCNTATCVLGRLADFLHRLQTYPRTNTGSNTY

SEQ ID NO：109 CNTATCVLGRLSQELHRLQTYPRTNTGSNT

SEQ ID NO：110 KCNTATCVLGRLSQELHRLQNFVPRTNTGSNTY

SEQ ID NO：111 KCNTATCVLGRLSQELHRLQTYPRTNTGSETF

SEQ ID NO：112 ACDTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：113 KCNTATCVLGRLSQELHRLQTYPRTNTGSKAP

SEQ ID NO：114 KCDTATCVTHRLAGLLSRSQTYPRTNTGSNTY

SEQ ID NO：115 KCNTATCVLGRLADALHRLQTYPRTNTGSNTY

SEQ ID NO：116 KCNTATCVLGRLAAFLHRLQTYPRTNTGSNTY

SEQ ID NO：117 SCNTATCVLGRLADFLHRLQTYPRTNTGSNTY

SEQ ID NO：118 KCNTATCVLGRLSQELHRLQTMPRTNTGSNTY

SEQ ID NO：119 KCNTATCVLGRLSQELHRLQTVPRTNTGSNTY

SEQ ID NO：120 KCNTATCVLGRLNEYLHRLQTYPRTNTGSNTY

SEQ ID NO：121 SCNTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：122 KCNTATCVLGRLTEFLHRLQTYPRTNTGSNTY

SEQ ID NO：123 KCNTATCVLGRLAEFLHRLQTYPRTNTGSNTY

SEQ ID NO：124 KCNTATCVLGRLTDYLHRLQTYPRTNTGSNTY

SEQ ID NO：125 KCNTATCVLGRLAQFLHRLQTYPRTNTGSNTY

SEQ ID NO：126 KCNTATCVLGRLADFLHRFQTFPRTNTGSNTY

SEQ ID NO：127 KCNTATCVLGRLADFLHRFHTFPRTNTGSNTY

SEQ ID NO：128 KCNTATCVLGRLADFLHRFQTFPRTNTGSGTP

SEQ ID NO：129 CNTATCVLGRLADFLHRLQTYPRTNTGSNTY

SEQ ID NO：130 KCDTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：131 KCNTATCVLGRLFDFLHRLQTYPRTNTGSNTY

SEQ ID NO：132 KCNTATCVLGRLAAALHRLQTYPRTNTGSNTY

SEQ ID NO：133 TCDTATCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：134 CSNLSTCATQRLANELVRLQTYPRTNVGSNTY

SEQ ID NO：135 KCNTATCATQRLANELVRLQTYPRTNVGSNTY

SEQ ID NO：136 CSNLSTCVLGRLSQELHRLQTYPRTNTGSNTY

SEQ ID NO：137 KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY

More on the one hand, compound of the present invention comprises the bioactive fragment of SEQ ID NO:40-137.Bioactive fragment can comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 or more a plurality of amino acid whose disappearance.In certain embodiments, the aminoacid sequence of SEQ IDNO:40-137 comprises at least 1,2,3,4,5,6,7,8,9,10 or more a plurality of modification, for example replaces, inserts, disappearance and/or derivation.In other embodiments, the aminoacid sequence of SEQ ID NO:40-137 has and is no more than 1,2,3,4,5,6,7,8,9 or 10 modification, for example replaces, inserts, lacks and or derivation.Of the present invention more on the one hand, compound of the present invention comprise with SEQ ID NO:40-137 in any have those compounds of at least 75,80,85,90,95 or 97% amino acid sequence identity.The identity percentage is by using Vector NTI (Invitrogen; Carlsbad CA) the AlignX module analysis in is measured.Meaning is applied to each independent SEQ ID NO with described each identity percentage or the bioactive fragment of mentioning or modification:.For example, described each embodiment, fragment, modification or identity percentage all are applicable to SEQ ID NO:40,41,42,43,44 etc., or arbitrary group of SEQ ID NO:.And compound of the present invention comprises the compound 1-127 that describes among the embodiment 3.

Another general aspect, compound of the present invention can play the agonist of at least a biological action of thyrocalcitonin disclosed herein, islet amyloid polypeptide and/or CGRP, or at least a in conjunction with in islet amyloid polypeptide, thyrocalcitonin or the CGRP acceptor.

Another general aspect, compound of the present invention can be used for reducing ingestion of food, reduce appetite, induce full sense, reduce the nutrition availability, cause weight loss, influence organization, change body energy content or energy expenditure, improve lipodogramme (comprise and reduce LDL cholesterol and triglyceride levels and/or change the HDL cholesterol levels), the digestive tract power that slows down, postpone stomach emptying, alleviate the postprandial blood sugar drift, prevent or glucagon suppression is secreted and brought high blood pressure down.

Therefore, in certain embodiments, method of the present invention is used for the treatment of or prevents by reduction relievable illness of nutrition availability or obstacle, comprises the The compounds of this invention that gives described experimenter's treatment or prevention significant quantity.Such illness and obstacle include but not limited to diabetes (comprising I type, II type and gestational diabetes), metabolic syndrome, dumping syndrome, hypertension, hyperlipemia, cardiovascular disorder, hyperlipidaemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and the osteoarthritis of eating disorder, insulin resistant, obesity, unusual postprandial hyperglycemia, any kind.

The limiting examples of cardiovascular disorder or disease is hypertension, myocardial ischemia and myocardial reperfusion.Compound of the present invention also can be used for treating or prevents and other fat relevant illness, comprise that apoplexy, cancer (for example carcinoma of endometrium, breast cancer, prostate cancer and colorectal carcinoma), gallbladder disease, sleep apnea, fertility descend and osteoarthritis, (referring to Lyznicki etc., Am.Fam.Phys.63:2185,2001).In other embodiments, compound of the present invention can be used for because of aesthetic former thereby change organization, to strengthen individual physical ability or to produce thinner meat source.

Another general aspect, compound of the present invention can be used for suppressing ghrelin secretion.Therefore, compound of the present invention can utilize this mechanism treatment or prevention ghrelin relative disease, for example Prader-Willi syndromes, all types of diabetes and complication thereof, obesity, hyperphagia, hyperlipidaemia or other disease relevant with supernutrition.

Another general aspect, It is generally accepted that now islet amyloid polypeptide and islet amyloid polypeptide agonist comprise compound of the present invention, can be used for treatment or prevention Barrett oesophagus, gastroesophageal reflux disease (GERD) and relative disease thereof.Such illness can include but not limited to pyrosis, stridulate with pyrosis, dysphagia, cough, intermittence that anti-influx of stomach/intestinal contents or lung accompany and vocal cords inflammation (with GERD relevant disease), oesophagus erosion, esophageal ulcer, esophagostenosis, Barrett metaplasia (substituting the normal esophageal epithelium with undesired epithelium), Barrett gland cancer and lung suck.Islet amyloid polypeptide and islet amyloid polypeptide agonist comprise The compounds of this invention, have the secretion inhibitor characteristic, for example gastric acid inhibitory, inhibition bile acide and inhibition pancreatin.And, have found that islet amyloid polypeptide has the stomach provide protection.Therefore, these characteristics of islet amyloid polypeptide, islet amyloid polypeptide agonist and The compounds of this invention can make it at Barrett oesophagus as described herein and/or GERD and relevant or follow in treatment of conditions and the prevention particularly useful.

Another general aspect, The compounds of this invention can be further used for the treatment or the prevention pancreatitis, carcinoma of the pancreas and gastritis.And The compounds of this invention is used in and has stood to treat among the patient of antidromicity courage pancreatic ductography (ERCP) under the endoscope and the prevention pancreatitis.Found that also islet amyloid polypeptide and islet amyloid polypeptide agonist (comprising The compounds of this invention) can have wonderful good therapeutic action when making up with somatostatin.Therefore, in certain embodiments, treatment or prevent pancreatitic method to comprise to give experimenter's islet amyloid polypeptide and islet amyloid polypeptide agonist (comprising The compounds of this invention), and give experimenter's somatostatin and somatostatin agonists.In other embodiments, treat or prevent pancreatitic method to comprise and give The compounds of this invention and give somatostatin and somatostatin agonists.

Another general aspect, The compounds of this invention also can be used for reducing bone resorption, reduces plasma calcium and brings out analgesic activity.Therefore, The compounds of this invention can be used for treating osteopathia, for example osteopenia and osteoporosis.In other embodiments, compound of the present invention can be used for treating pain and painful neuropathy.

Another general aspect, compound of the present invention can be used as the part of combination therapy.In certain embodiments, compound of the present invention can with other commercially available meals auxiliary that gets or other antiobesity agent coupling, as an example, antiobesity agent for example is PYY and PYY agonist, GLP-1 and GLP-1 agonist, DPPIV inhibitor, CCK and CCK agonist, Exendin and exendin peptide agonists, and Leptin and Leptin agonist.In other embodiments, compound of the present invention can with other analgesic agent, immunosuppressor or other antiphlogiston coupling.

Another general aspect, the novel pharmaceutical compositions and the using method thereof that contain The compounds of this invention have been described.In certain embodiments, medicinal compositions can comprise 0.01%-5%w/v at least.In certain embodiments, the pH of composition can be about 3.0 to about 6.0.In certain embodiments, damping fluid can be acetate, phosphoric acid salt, Citrate trianion or glutaminate.In certain embodiments, composition further comprises carbohydrate or polyvalent alcohol tonicity agents (tonicifier).In certain embodiments, composition further comprises sanitas, and it is selected from: meta-cresol, benzylalcohol, p-Hydroxybenzoate and phenol.In other embodiments, give described compound to the dosage of about 2mg/kg with about 0.05mg/kg.For example, per daily dose can be every day 1 μ g to about 5mg.In certain embodiments, the representational prescription formula of passing can be injection, infusion, absorption (mucous membrane) and suction.Route of administration can be intramuscular, intravenously, subcutaneous, through skin, in mucous membrane, oral, nose or by the lung inhalation.

Also imagination will encode this paper describe aminoacid sequence Nucleotide, contain described Nucleotide carrier, be used for amplification of nucleotide and/or express the coded polypeptide of Nucleotide host cell, at the antibody of novel cpd and the experimenter screen or the purposes of detection/diagnosis illness as described herein as a part of the present invention.

Can more be expressly understood these aspects of the present invention and others with reference to following preferred embodiment and detailed description.

The accompanying drawing summary

Fig. 1 has shown that food consumption is to the dose-dependently decline (ingestion of food experiment) of representative compounds dosage of the present invention in the mouse of overnight fasted.

Fig. 2 A illustrates the calorie intake that continues periphery infusion solvent or representative compounds of the present invention in getting fat (meals inducibility obesity or DIO) rat in 2 cycles to be reduced.

Fig. 2 B illustrates the decline of body weight in the corresponding time cycle.

Fig. 3 has illustrated the effect of representative compounds of the present invention to organization.

Fig. 4 A has illustrated the effect of representative compounds of the present invention to stomach emptying.

Fig. 4 B has illustrated the low blood calcium effect of representative compounds of the present invention.

Fig. 5 has illustrated the effect of representative compounds of the present invention to triglyceride level.

Fig. 6 A-6B has illustrated the effect of time dependent representative compounds of the present invention to triglyceride level.

Fig. 7 A-7E has illustrated the dosage effect of time dependent representative compounds of the present invention to triglyceride level.

Fig. 8 A has illustrated the effect of islet amyloid polypeptide to ghrelin.

Fig. 8 B-8C has illustrated the effect of islet amyloid polypeptide to ghrelin.

Fig. 9 A has illustrated the effect of representative compounds of the present invention to the pancreas function marker.

Fig. 9 B has illustrated the effect of representative compounds of the present invention to the pancreas function marker.

Figure 10 A has illustrated the effect of islet amyloid polypeptide to gastric acid secretion.

Figure 10 B has illustrated the dose response effect of islet amyloid polypeptide to gastric acid secretion.

Figure 11 A has illustrated the stomach provide protection of islet amyloid polypeptide.

Figure 11 B has illustrated the stomach provide protection of islet amyloid polypeptide.

Figure 11 C has described the dose response curve of islet amyloid polypeptide.

Detailed Description Of The Invention

The present invention relates to novel amylin family compound or agonist (being also referred to as novel cpd and The compounds of this invention).These novel cpds can be used for treating or preventing the illness of metabolic trouble, vascular disease, ephrosis and/or gastrointestinal illness and so on.Previous chapters and sections provide the structure of compound.Novel cpd can further be described to have required feature, for example has with islet amyloid polypeptide, thyrocalcitonin and/or CGRP with obstacle and above-mentioned those treatment of diseases and/or in preventing at metabolic trouble and compares equal or higher activity.In other embodiments, compare with islet amyloid polypeptide, thyrocalcitonin or CGRP, compound of the present invention can not have equal or higher activity, but can have biological activity, active outbreak faster, the long active time-histories of the stability of increase and solvability, less side effect, combination and/or be easy to produce, preparation or use.

So-called islet amyloid polypeptide activity refers to that compound shows the physiological characteristic that is similar to islet amyloid polypeptide, and those physiological characteristics of for example describing in this specification sheets for example reduce ingestion of food.The compounds of this invention can interact with it with islet amyloid polypeptide acceptor or islet amyloid polypeptide self and excite other one or more receptors bind of biological respinse (for example reducing ingestion of food) or otherwise directly or indirectly interaction.

So-called thyrocalcitonin activity refers to that compound shows the physiological characteristic that is similar to thyrocalcitonin, and those physiological characteristics of for example describing in this specification sheets for example suppress the osteoclast function.The compounds of this invention can interact with it with CT acceptor or thyrocalcitonin self and excite other one or more receptors bind of biological respinse (for example being suppressed to bone cell function) or otherwise directly or indirectly interaction.

So-called CGRP activity refers to that compound shows the physiological characteristic that is similar to CGRP, and those physiological characteristics of for example describing in this specification sheets for example excite vasorelaxation action.Compound of the present invention can interact with it with CGRP acceptor or CGRP self and excite other one or more receptors bind of biological respinse (for example exciting vasorelaxation action) or otherwise directly or indirectly interaction.

The compounds of this invention can also comprise possessing of larger peptide described herein of active bioactive fragment.Therefore, the required active example that The compounds of this invention has comprises that (1) has the activity that is similar to islet amyloid polypeptide, thyrocalcitonin or CGRP in ingestion of food, stomach emptying, pancreatic secretion, blood pressure, heart rate or the experiment that loses weight, and/or (2) in receptors bind experiment in conjunction with islet amyloid polypeptide, thyrocalcitonin or CGRP.Some representative experiment is provided in embodiment 1.

The compounds of this invention can further have specific binding pattern.For example, reported already, by means of combine the biological effect of mediation islet amyloid polypeptide, thyrocalcitonin and CGRP with two kinds of closely-related II type g protein coupled receptors (GPCR)-Calcitonin Receptor (CTR) and Calcitonin Receptor sample acceptor (CRLR).Clone and functional study show the interaction of CGRP and islet amyloid polypeptide and CTR or CRLR and receptor active modified protein (RAMP) various combination.The multiple RAMP of many cell expressings.It is generally acknowledged that the coexpression of RAMP and CTR or CRLR is that generation thyrocalcitonin, CGRP and islet amyloid polypeptide functional receptor are necessary.RAMP family comprises 3 members (RAMP-1 ,-2 and-3), and its sequence identity of enjoying is lower than 30%, but has common topology group structure.CRLR and RAMP1 coexpression cause forming the CGRP acceptor, and CRLR and RAMP3 coexpression also cause forming the CGRP acceptor.HCTR2 and RAMP1 coexpression cause forming islet amyloid polypeptide and CGRP acceptor.HCTR2 and RAMP3 coexpression cause forming the islet amyloid polypeptide acceptor.

Therefore, the The compounds of this invention that is used for the inventive method can show the affinity with islet amyloid polypeptide, CGRP and the Calcitonin Receptor of amylin family.The compounds of this invention can demonstrate the significant affinity in conjunction with the islet amyloid polypeptide acceptor, and in conjunction with the ability of other acceptor (for example thyrocalcitonin and CGRP acceptor).The compounds of this invention can be higher than the affinity of 20nM, 10nM, 5nM, 1nM in conjunction with the islet amyloid polypeptide acceptor, more preferably with the affinity that is higher than 0.10nM in conjunction with the islet amyloid polypeptide acceptor.In addition, the affinity that The compounds of this invention can also be similar is in conjunction with thyrocalcitonin and CGRP acceptor, but lower with the affinity of CGRP acceptor.In other embodiments, The compounds of this invention can be higher than the affinity of 20nM, 10nM or 1nM in conjunction with Calcitonin Receptor.In other embodiments, The compounds of this invention can be higher than the affinity of about 1 μ M, 700nM or 500nM in conjunction with the CGRP acceptor.The compounds of this invention also can be in conjunction with all three kinds of acceptors to different degree.Therefore, the imagination The compounds of this invention can have the bind profile that each acceptor of hormone described herein is had specific affinity.

With regard to reduce that ingestion of food and this paper (for example table 1) describes a kind of other active with regard to, The compounds of this invention comprises those compounds of formula I, II, III, IV; The bioactive fragment of SEQ ID NO:40-137; Have those compounds and the bioactive fragment thereof of at least 75%, 80%, 82%, 85%, 87%, 90%, 92%, 95% or 97% amino acid sequence identity with any of SEQ ID NO:40-137, can possess islet amyloid polypeptide, thyrocalcitonin, CGRP or have the compound of SEQ ID NO:40-137 sequence or the compound 1-137 among the embodiment 3 at least about 25% biological activity, preferred about biological activity of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%.In another embodiment, The compounds of this invention has the biological activity of improvement.Preferably, with regard to reduce that ingestion of food and this paper (for example table 1) describes a kind of other active with regard to, novel cpd has islet amyloid polypeptide, thyrocalcitonin, CGRP or has the compound of SEQ ID NO:40-137 sequence or the compound 1-137 among the embodiment 3 bioactive at least about 110%, 125%, 130%, 140%, 150%, 200% or higher.For example, required The compounds of this invention is that the activity that is had in a kind of experiment described herein (ingestion of food, the experiment that loses weight, stomach emptying, triglyceride level, pancreatitis, ghrelin or calcium) is higher than islet amyloid polypeptide, thyrocalcitonin or the CGRP active compound in identical experiment.

Only as example, required The compounds of this invention can show the ability that reduces the accumulation ingestion of food, and it is compared with solvent, and it reduces to surpass 5%, preferably surpasses 15%, more preferably surpasses 25%, more preferably surpasses 35% or 40% again, most preferably surpasses 50%.

Another general aspect, The compounds of this invention can be used for reducing ingestion of food, reduce appetite, induce full sense, reduce the nutrition availability, cause weight loss, influence organization, change body energy content or energy expenditure, improve lipodogramme (comprise and reduce LDL cholesterol and triglyceride levels and/or change the HDL cholesterol levels), the digestive tract power that slows down, postpone stomach emptying, alleviate the postprandial blood sugar drift, prevent or glucagon suppression is secreted and brought high blood pressure down.At least in embodiment 3,4,5,6 and 7, described about to ingestion of food, lose weight, the representativeness experiment of stomach emptying, triglyceride level and organization effect.

Therefore, in certain embodiments, the inventive method is used for the treatment of or prevents by reduction relievable illness of nutrition availability or obstacle, comprises the The compounds of this invention that gives described experimenter's treatment or prevention significant quantity.Such illness and obstacle include but not limited to diabetes (comprising I type, II type and gestational diabetes), metabolic syndrome, dumping syndrome, hypertension, hyperlipemia, cardiovascular disorder, hyperlipidaemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and the osteoarthritis of eating disorder, insulin resistant, obesity, unusual postprandial hyperglycemia, any kind.

The limiting examples of cardiovascular disorder or disease is hypertension, myocardial ischemia and myocardial reperfusion.The compounds of this invention also can be used for treating or prevents and other fat relevant illness, comprise that apoplexy, cancer (for example carcinoma of endometrium, breast cancer, prostate cancer and colorectal carcinoma), gallbladder disease, sleep apnea, fertility reduce and osteoarthritis, (referring to Lyznicki etc., Am.Fam.Phys.63:2185,2001).In other embodiments, The compounds of this invention can be used for because of aesthetic former thereby change organization, to strengthen individual physical ability or to produce thinner meat source.

Another general aspect, The compounds of this invention can be used for suppressing ghrelin secretion.Therefore, The compounds of this invention can utilize this mechanism treatment or prevention ghrelin relative disease, for example Prader-Willi syndromes, all types of diabetes and complication thereof, obesity, hyperphagia, hyperlipidaemia or other disease relevant with supernutrition.Representativeness experiment to the ghrelin effect is described in embodiment 8.

Another general aspect, It is generally accepted that now islet amyloid polypeptide and islet amyloid polypeptide agonist comprise The compounds of this invention, can be used for treatment or prevention Barrett oesophagus, gastroesophageal reflux disease (GERD) and relative disease thereof.Such illness can include but not limited to pyrosis, stridulate with pyrosis, dysphagia, cough, intermittence that anti-influx of stomach/intestinal contents or lung accompany and vocal cords inflammation (with GERD relevant disease), oesophagus erosion, esophageal ulcer, esophagostenosis, Barrett metaplasia (substituting the normal esophageal epithelium with undesired epithelium), Barrett gland cancer and lung suck.Islet amyloid polypeptide and islet amyloid polypeptide agonist comprise The compounds of this invention, have the secretion inhibitor characteristic, for example gastric acid inhibitory, inhibition bile acide and inhibition pancreatin.Therefore, these characteristics of islet amyloid polypeptide, islet amyloid polypeptide agonist and The compounds of this invention can make it at Barrett oesophagus as described herein and/or GERD and relevant or follow in treatment of conditions and the prevention particularly useful.Demonstration is described in embodiment 10 and 11 to the effect of gastric acid secretion and the representativeness experiment of stomach provide protection.

Another general aspect, The compounds of this invention can be further used for the treatment or the prevention pancreatitis, carcinoma of the pancreas and gastritis.And The compounds of this invention is used in treatment and prevention pancreatitis among the patient who stands antidromicity courage pancreatic ductography (ERCP) under the endoscope.Found that also islet amyloid polypeptide and islet amyloid polypeptide agonist (comprising The compounds of this invention) can have wonderful good therapeutic action when making up with somatostatin.Therefore, in certain embodiments, treatment or prevent pancreatitic method to comprise to give experimenter's islet amyloid polypeptide and islet amyloid polypeptide agonist (comprising The compounds of this invention), and give experimenter's somatostatin and somatostatin agonists.In other embodiments, treat or prevent pancreatitic method to comprise and give The compounds of this invention and give somatostatin and somatostatin agonists.Demonstration is described in embodiment 9 to the representativeness experiment of the effect of pancreas function.

Another general aspect, compound of the present invention also can be used for reducing bone resorption, reduces plasma calcium and brings out analgesic activity.Therefore, The compounds of this invention can be used for treating osteopathia, for example osteopenia and osteoporosis.In other embodiments, The compounds of this invention can be used for treating pain and painful neuropathy.Demonstration is provided in embodiment 6 to the representativeness experiment of calcium level effect.

In the methods of the invention, described polypeptide can (include but not limited to contain islet amyloid polypeptide or islet amyloid polypeptide analogue agonist to other compound and composition that reduction nutrition availability has long-acting or fugitive effect individually or with one or more, salmon calcitonin see calcimar, cholecystokinin (CCK) or CCK agonist, Leptin (OB albumen) or Leptin agonist, Exendin or exendin peptide analogs agonist, GLP-1 or GLP-1 analogue agonist, the DPPIV inhibitor, PYY or PYY analogue, AFP-6 (intermedin) or AFP-6 agonist, other compound and the composition of Urocortin or Urocortin agonist or adrenomedullin or adrenomedullin agonist) give together.Suitable islet amyloid polypeptide agonist for example comprises [25,28,29 Pro-] people's islet amyloid polypeptide (be also referred to as " tripro-amylin ", be set forth in United States Patent (USP) the 5th, 686,511 and 5,998, No. 367).The CCK that uses is preferably CCK octapeptide (CCK-8).Leptin for example is discussed in (Pelleymounter, Cullen etc., Science 269:540-543 (1995); Halaas, Gajiwala etc., Science 269:543-6 (1995); Campfield, Smith etc., Science 269:546-549 (1995)).Suitable Exendin comprises Exendin-3 and Exendin-4, and the Exendin agonist compound comprises those compounds that for example are described in PCT prospectus WO 99/07404, WO99/25727 and WO 99/25728.Suitable PYY polypeptide and analogue comprise and are described in U. S. application the: those compounds of [proxy's application case numbering 18528.662 and 18528.663].

Surpass 30 although " obesity " is commonly defined as body-mass index, concerning this specification sheets, need or wish slimming arbitrary experimenter, comprise that body-mass index is lower than those experimenters of 30, all is included in " obesity " scope.

The preparation of The compounds of this invention

Can use standard recombinant technology known in the art or chemical peptide synthetic technology (for example using automatization or semi-automatic peptide synthesizer), or these two, prepare The compounds of this invention described herein.Equally, can use method in standard chemical, biological chemistry or the body, the derivative of production polypeptide of the present invention.

Can be according to routine techniques, synthetic The compounds of this invention in solution or on the solid support.The commercially available acquisition of various automatization synthesizers, and can use in accordance with known methods.Referring to for example Stewart and Young, Solid Phase Peptide Synthesis, the 2nd edition, PierceChemical Co. (1984); Tam etc., J.Am.Chem.Soc.105:6442 (1983); Merrifield, Science 232:341-7 (1986); And Barany and Merrifield, ThePeptides, Gross and Meienhofer edit, Academic Press, New York, 1-284 (1979).The automated peptide synthesizer of available use NMP/HOBt (option one) system (430A type for example, Applied Biosystems Inc., Foster City, California) and comprise the tBoc that adds cap or the Fmoc chemical method (guides referring to the AppliedBiosystems user about ABI 430A peptide synthesizer, 1.3B version, on July 1st, 1988, the 6th joint, 49-70 page or leaf, Applied Biosystems, Inc., Foster City California) carries out solid-phase peptide and synthesizes.Also can use Advanced ChemTech synthesizer (MPS 350 types, Louisville, Kentucky) assembled peptide.Can use for example Waters Delta Prep 3000 systems and C4, C8 or C18 preparative column (10 μ, 2.2 * 25cm; Vydac, Hesperia, California), by RP-HPLC (preparation type and analysis mode) purified peptide.Easily composite reactive albumen screens activated protein with the screening experiment that is designed for the identification peptide then.

Except classics by the step synthetic, developed the property compiled (convergent) solid-phase peptide synthetic (being also referred to as hybrid method or fragment condensation), be used to prepare peptide and small protein complicated and difficulty.According to this method, perhaps on solid support, perhaps in solution, the suitable shielded peptide fragment that covers whole peptide sequence and prepare on solid phase is condensed into target peptide.The utilizability of new resin and plastic resin treatment has been opened the possibility by the fast synthetic complete shielded peptide sections of solid phase technique.This method is attractive especially to producing macromole, because it has made up the two advantage of solid phase and liquid phase method.Production cycle is shorter than liquid phase process, and output and purity are higher usually.In addition, scale-up is easier, has avoided many rendezvous problems that often run in long peptide solid phase synthesis.

In another embodiment, synthesis strategy uses and compiles fragment condensation.Compile fragment condensation and be and a kind ofly be combined to the method for comparing the more superior big peptide of production high quality with standard solid-phase or reference liquid.Use such method can parallel synthetic fragment, reduction generated time and guarantee quality.Along with peptide becomes long more, side reaction and incomplete synthetic risk are just high more.Confirmed already that peptide sequence of the present invention can have the glycine and the proline(Pro) of desired position, it has facilitated the fragmentation method, because this two seed amino acid can not racemize in fragment coupling process, and demonstrates very effective condensation rate.Except therefore the bigger amplification production problem of direct solid phase synthesis existence also may be unsuitable for the extensive synthetic fact, the fragmentation method was more controlled, and the chance of purify intermediates is provided.Disulfide linkage only is arranged in first fragment, and therefore can form with complete shielded precursor in the fragment stage.Because there are a lot of advantages in this method aspect strategy and the scale, thus generated time will be shortened, because all fragments all can be synthesized simultaneously.This is a perfect way that reduces risk to greatest extent and reduce cost.For showing the desirable segmental skeleton structure that can allow this strategy execution, following fragment has been described:

Fragment 1:Boc-X (Boc)-X-X (Trt)-X (tBu)-X-X (tBu)-X-X-X-Gly-OH

Ring-type (cyclisation should occur in before the fragment coupling)

Fragment 2:Fmoc-K (Pbf)-X-X (tBu)-X (Trt)-X (OtBu)-X-X (Trt)-X (Pbf)-X-X (Trt)-X (tBu)-X (tBu)-Pro-OH

Fragment 3:Fmoc-X (Pbf)-X (tBu)-X (Trt)-X (tBu)-X-X (tBu)-X (Trt)-Thr (tBu)-OH

Fragment 4:H-Tyr (tBu)-NH ₂

Tell about according to these, which The compounds of this invention those skilled in the art can understand, and to be used for this synthetic should be ideal.Constantly the technology of development makes and only has 3 fragments and become possibility, and promptly the 4th fragment do not need to be independent of the 3rd fragment and produce.

On the other hand, can produce The compounds of this invention by recombinant technology well-known in the art.Referring to for example Sambrook etc., Molecular Cloning:A Laboratory Manual, the 2nd edition, Cold Spring Harbor (1989).Can express by polynucleotide by these polypeptide that recombinant technology produces.These polynucleotide sequences can be mixed with and be beneficial to the codon that mRNA transcribes and translates in microorganism host.According to method well-known in the art, can easily make up the sequence of production like this.Referring to for example WO 83/04053.The above polynucleotide N end methionyl residue of also can randomly encoding.Can utilize the methods known in the art preparation to be used for non-peptide compound of the present invention.For example, can use methods known in the art to prepare the amino acid of phosphoric acid and contain these amino acid whose peptides.Referring to for example Bartlett and Landen, BioorgChem.14:356-77 (1986).

Can utilize various expression vector/host systems, to comprise and to express the novel cpd encoding sequence.These systems include but not limited to microorganism, for example the bacterium that transforms with recombinant phage, plasmid or cosmid DNA expression vector; Yeast with the Yeast expression carrier conversion; Insect cell system with virus expression carrier (for example baculovirus) infection; With virus expression carrier (cauliflower mosaic virus CaMV for example; Tobacco mosaic virus (TMV) TMV) transfection or with bacterial expression vector (for example Ti or pBR322 plasmid) plant transformed cell system; Or zooblast system.The mammalian cell that is used for recombinant protein production includes but not limited to that VERO cell, HeLa cell, Chinese hamster ovary (CHO) clone, COS cell (for example COS-7), WI 38, BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562 know 293 cells.Hereinafter described and be used for the recombinant expressed representative solution of albumen.

Therefore, polynucleotide sequence provided by the invention can be used for producing the incubation growth method of new and useful virus and plasmid DNA carrier, new and useful conversion and transfection protokaryon and eukaryotic host cell (being included in bacterium, yeast and the mammalian cell of growing in the culture) and new and useful this host cell that can express polypeptide of the present invention.The polynucleotide sequence of the novel cpd of coding this paper also can be used for gene therapy.

The present invention also provides the recombinant DNA production method of novel cpd.The method of being produced polypeptide by the host cell that contains the novel cpd coding nucleic acid is provided, and it comprises: (a) cultivate the described host cell that contains these these peptide coding polynucleotide helping expressing under the condition of this dna molecular; (b) obtain described novel cpd.

Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, comprises bacterium, mammalian cell (for example Chinese hamster ovary (CHO) cell, MC, baby hamster kidney cell, cancer cells or other cell), yeast cell and insect cell.

The mammalian hosts system that is used for express recombinant protein also is well-known for a person skilled in the art.Can select host cell strain according to processing expressing protein or producing the certain capabilities that some is used to provide the posttranslational modification of protein-active.Peptide modified acetylize, carboxylation, glycosylation, phosphorylation, fatization and the acidylate of including but not limited to like this.Cutting " preceding former " form proteic translation post-treatment may be also very important to correct insertion, folding and/or function.Different host cells, for example CHO, HeLa, MDCK, 293, WI38 etc. have specialized cell machine and feature mechanism to activity after such translation, can host cell be selected the correct modification and the processing of the foreign protein that is imported to guarantee.

Perhaps, can use Yeast system to produce novel cpd of the present invention.Coding region by pcr amplification novel cpd coding DNA.Use a kind of Nucleotide 1-20 that contains α mating type factor gene primer and with the another kind of primer of the Nucleotide 255-235 complementary of this gene, in PCR reaction by pastoris genomic dna amplification coding yeast before-DNA (Kurjan and the Herskowitz of former-α leader sequence, Cell, 30:933-43 (1982)).Preceding-former-α pilot code sequence and novel cpd encoding sequence fragment are connected in the plasmid that contains yeast alcohol dehydrogenase (ADH2) promotor, make the guiding of this promotor by preceding-the Expression of Fusion Protein that former-alpha factor is formed that merges to ripe novel cpd.As Rose and Broach, Meth.Enz.185:234-79, Goeddel edits, Academic Press, Inc., San Diego, California (1990) tells about, and carrier further comprises ADH2 transcription terminator, yeast " 2-μ " replication orgin, yeast leu-2d gene, yeast REP1 and REP2 gene, intestinal bacteria β-Nei Xiananmei gene and the intestinal bacteria replication orgin in cloning site downstream.β-Nei Xiananmei and leu-2d gene are respectively for selecting in bacterium and yeast.The leu-2d gene also helps increases the copy number of plasmid in yeast, to induce the expression of higher level.REP1 and REP2 genes encoding participate in regulating the albumen of plasmid copy number.

Use currently known methods, for example lithium acetate is handled (Steams etc., Meth.Enz.185:280-97 (1990)), and the DNA construction that will describe in leading portion is transformed in the yeast cell.When exhausting, the glucose in the growth medium induces ADH2 promotor (Price etc., Gene 55:287 (1987)).Preceding-former-α sequence influences the emiocytosis fusion rotein.What accompany therewith is that yeast KEX2 albumen cuts preceding-former sequence (Bitter etc., Proc.Natl.Acad.Sci.USA 81:5330-4 (1984)) from maturing compound of the present invention.

Can also use commercially available expression system, for example pichia spp (Pichia) expression system (Invitrogen, San Diego, California), according to manufacturer's explanation, recombinant expressed The compounds of this invention in yeast.Before this system also relies on-and former-α sequence guiding secretion, drive by alcohol oxidase (AOX1) but insert segmental transcribing when methanol induction.By for example being used for method, by the secreted novel cpd of yeast growth substratum purifying by bacterium and mammalian cell supernatant liquor purifying novel cpd.

Perhaps, the dna clone of encoding novel compound can be gone into rhabdovirus expression vector pVL1393 (PharMingen, San Diego, California) in.According to manufacturer's guide (PharMingen), noctuid (Spodoptera frugiperda) cell is coveted on the meadow of using the carrier that contains this novel cpd to infect in the no sF9 protein culture medium then, and produces recombinant protein.(Amicon, Beverly Massachusetts) by purifying in the substratum and protein concentrate, and are resuspended among the PBS molecular sieve column that uses heparin-Sepharose post (Pharmacia, Piscataway, New Jersey) and be connected in series.SDS-PAGE analyzes and shows single band, and conclusive evidence albumen size is carried out the Edman order-checking with Proton 2090 peptide sequencers and confirmed its N terminal sequence.

For example, the DNA sequences encoding of required novel cpd can be cloned in the plasmid that contains required promotor and optional leader sequence (referring to for example Better etc., Science 240:1041-3 (1988)).The sequence of this construction can be confirmed by the automatization order-checking.Use then and adopt CaCl ₂The standard method that incubation and bacterium heat shock are handled is transformed into (Sambrook etc., ibid) among the coli strain MC1061 with plasmid.The bacterium that transforms is cultivated in adding the LB substratum of Pyocianil, produces by growth inducing expressing protein in suitable medium.If there is leader sequence, then leader sequence will influence the secretion of ripe novel cpd, and cut in secretion process.Utilize method described below by recombinant protein purifying secreted in the bacteria culture medium.

Perhaps, polypeptide of the present invention can be expressed in the insect system.The insect system that is used for protein expression is that those skilled in the art are known.In such system, use the carrier of autographa california nuclear polyhedrosis virus (AcNPV) as expression alien gene in greedy frugiperda cell in meadow or powder Noctua (Trichoplusia) larva.The novel cpd encoding sequence is cloned in the nonessential zone (for example polyhedron gene) of virus, and is under the control of polyhedrin promotor.The successful insertion of novel cpd will make the polyhedron gene inactivation, produce the recombinant virus that does not have coat protein bag quilt.Use the recombinant virus infection wherein greedy frugiperda cell in meadow or powder Noctua larva (Smith etc., the J.Virol.46:584 (1983) of express polypeptide then; Engelhard etc., Proc.Natl.Acad.Sci.USA 91:3224-7 (1994)).

In another embodiment, can pass through the dna sequence dna of pcr amplification encoding novel compound, and it is cloned in the suitable carriers, for example pGEX-3X (Pharmacia, Piscataway, New Jersey).The pGEX carrier design is used to produce fusion rotein, and it comprises by the glutathione-S-transferase of vector encoded (GST) with by the dna fragmentation encoded protein that is inserted in the carrier cloning site.Can produce the PCR primer that contains for example suitable cleavage site.Can partly cut recombination fusion protein by the GST of fusion rotein then.PGEX-3X/PYY analogue polypeptide construction is transformed into intestinal bacteria XL-1 Blue cell (Stratagene, La Jolla, California) in, separate single transformant, and in LB substratum (adding Pyocianil), be 0.4 in 37 ℃ of optical density(OD) that are cultured to the 600nm wavelength, then (Missouri) there are down incubation 4 hours in Sigma Chemical Co., St.Louis again at 0.5mM isopropyl ss-D-thio-galactose pyran-glucoside.The plasmid DNA of purification of individual transformant, and use the automatization sequenator partly to check order, to confirm the existing goal gene of correct direction to insert fragment.

Being expected at the fusion rotein that can be used as insoluble inclusion body production in the bacterium can following purifying.Pass through centrifugal collecting cell; Clean with 0.15M NaCl, 10mM Tris (pH8), 1mMEDTA; And with 0.1mg/mL N,O-Diacetylmuramidase (Sigma Chemical Co.) in room temperature treatment 15 minutes.Supersound process clarification lysate is by with centrifugal 10 minutes sedimentation cell fragments of 12,000 * g.The pellet resuspended that will contain fusion rotein is layered on 50% glycerine in 50mM Tris (pH8) and 10mM EDTA, and with 6000 * g centrifugal 30 minutes.Pellet resuspended is at no Mg ⁺⁺And Ca ⁺⁺Standard phosphate buffered saline(PBS) (PBS) in.Be further purified fusion rotein (Sambrook etc., ibid) by the precipitation of fractional separation resuspension in the sex change sds page.Gel is immersed among the 0.4M KCl, manifesting albumen, downcuts albumen, and in not having the gel-runing buffer of SDS electroelution.If as soluble proteins production, then it can use GST purifying module (GST Purification Module) (Pharmacia Biotech) purifying to GST/ novel cpd fusion rotein in bacterium.

Fusion rotein can digest, to cut GST and ripe novel protein.(20-40 μ g fusion rotein, 20-30 unit's human thrombin (the 0.5mL PBS solution of 4000U/mg (Sigma)) be in room temperature incubation 16-48 hour, and go up sex change SDS-PAGE gel, with the fractional separation reaction product for the digestion reaction thing.Gel is immersed among the 0.4M KCl, to manifest protein band.(Applied Biosystems 473A type, Foster City California), by the partial amino-acid series analysis, can confirm the identity corresponding to the protein band of novel cpd expection molecular weight to use the automatization sequenator.

In particularly preferred novel cpd recombinant expression method, can pass through calcium phosphate method, be used in plasmid co-transfection 293 cells that contain novel cpd DNA among pCMV carrier (5 ' CMV promotor, 3 ' HGH polyadenylic acid sequence) and the pSV2neo (containing the neo resistant gene).Preferably, carrier should be used the ScaI linearizing before transfection.Equally, can use substituting construction, its use is mixed with the similar pCMV carrier of neo gene.By in the growth medium that contains 0.5mg/mL G418 (Xin Meisu sample microbiotic) limiting dilution 10-14 days, select stable cell lines by single cell clone.By ELISA or western blotting, pair cell system carries out the novel cpd expression screening, and amplification high expressing cell system, to be used for extensive growth.

Preferably transformant is used for for a long time, the protein production of high yield, therefore need stably express.Transform such cell in case use the carrier that contains selective marker and required expression cassette, just allow cell in enrichment medium, grow 1-2 days, then it is converted to the selection substratum.Selective marker is used to give the selection resistance, and its existence makes the cell of sequence that successful expression imports to grow and to reclaim.Can use the resistance group (Resistant clumps) of the tissue culture technique propagation stable transformed cells that is suitable for this cell.

Can use many selective systems to reclaim and transform and be used for the cell that recombinant protein is produced.Such selective system includes but not limited to the HSV thymidine kinase in tk-, hgprt-or aprt-cell, xanthoglobulin-guanine phosphoribosyltransferase and adenine phosphoribosyl transferase gene respectively.In addition, the metabolic antagonist resistance can be used as the selection basis of dhfr, gpt, neo, also and hygro, and dhfr gives the resistance to methotrexate; Gpt gives the resistance to mycophenolic acid; Neo gives the resistance to aminoglycoside G418; Also gives the grand resistance of chlorine sulphur; And hygro gives the resistance to Totomycin.Other spendable selection gene comprises trpB or hisD, and trpB allows cell to utilize indoles to replace tryptophane, and hisD allows cell to utilize histidinol to replace Histidine.Generation is used to differentiate that the mark of the range estimation indication of transformant comprises anthocyanin, beta-Glucuronidase and substrate GUS thereof and luciferase and substrate fluorescein thereof.

Can use the two combinations produce multiple novel cpd of the present invention of the synthetic and recombinant technology of automated peptide.For example, compound of the present invention can be included in the modification combination before the PEGization, comprises disappearance, displacement and insertion.Such compound can be produced stage by stage.In the fs, can contain the novel cpd of intermediate forms of the modification of disappearance, displacement, insertion and arbitrary combination thereof by recombinant technology production as described.Then, behind optional purification step as described below, by (for example deriving from Nectar TransformingTherapeutics, San Carlos with suitable PEGization reagent, California) by chemically modified PEGization intermediate polypeptide, to produce required compound.Those of skill in the art will recognize that above-mentioned steps can be applied to contain the novel cpd of modifying combination, described modification is selected from other modification means that disappearance, displacement, insertion, derivation and this area are well-known and the present invention imagines.

The novel cpd that may need purifying the present invention to produce.The peptide purification technology is that those skilled in the art are well-known.It is polypeptide and non-polypeptide fraction that these technology are included on the level the cellular environment roughing out.After with described polypeptide and other albumen sepn, can use chromatography and electrophoretic technique to be further purified desired polypeptides, to reach partially or completely purifying (or being purified to homogeneous).The analysis mode method that is particularly suitable for pure peptide preparation is ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis and isoelectrofocusing.Especially effectively the peptide purification method is a reversed-phase HPLC, then characterizes purified product by liquid chromatography/mass spectrometry method (LC/MS) and substance assistant laser desorpted ionization (MALDI) mass spectroscopy.By determining that amino acid analysis obtains the extra confirmation to purity.

Some aspect of the present invention relates to the purifying of coded albumen or peptide, in specific embodiment, relates to the basic purifying of coded albumen or peptide.The various technology that are applicable to peptide purification are that those skilled in the art are known.These technology comprise precipitations such as for example using ammonium sulfate, PEG, antibody; Thermally denature, then centrifugal; Chromatographic step, for example ion exchange chromatography, gel permeation chromatography, reversed phase chromatography, hydroxyapatite and affinity chromatography; Isoelectrofocusing; Gel electrophoresis; And the combination of these technology and other technology.As this area, known, it is believed that to change the order of carrying out various purification steps, or some step can omit, still can form the method for the albumen or the peptide that are suitable for preparing basic purifying.The method of purified polypeptide is found in United States Patent (USP) the 5th, 849, No. 883.These file descriptions the particular representative method of separation and purifying G-CSF composition, these methods can be used for separating and purifying novel cpd of the present invention.Under the situation of the known disclosure of invention, those skilled in the art obviously can be appreciated that and can use numerous purification techniques by given source purified polypeptide.In addition, imagination can be used the combinations produce purifying compounds of the present invention of anionresin and immunoaffinity chromatography.

Therefore, phrase " isolated polypeptide or peptide " refers to the proteic polypeptide of other impurity or the peptide in essentially no cellular material or dietary protein origin cell or tissue source, or when chemosynthesis the polypeptide or the peptide of essentially no precursor or other chemical substance.Statement " essentially no cellular material " comprises that wherein said albumen and separation or reorganization produce the isolating protein product of cellular component of its cell.Therefore, the albumen of acellular substantially material comprises that heterologous protein (being also referred to as " impurity albumen " at this paper) is lower than the protein product of about 30%, 20%, 10% or 5% (dry weight).When albumen, peptide and fragment reorganization thereof produced, preferably it did not also have substratum substantially, promptly cultivates not enough about 20%, 10% or 5% of fiduciary point protein product volume.When albumen produced by chemosynthesis, preferably it did not have precursor or other chemical substance substantially, and promptly it separates with participation albumen synthetic precursor or other chemical substance.Therefore, the compound beyond the precursor of such protein product or the desired polypeptides is lower than about 30%, 20%, 10%, 5% (dry weight).In preferred embodiments, the not general any impurity albumen of producing proteic same animals of purifying or isolating goods, this can by for example in inhuman cell recombinant expressed people's albumen finish.

Some preferred synthetic method is described in asks the 454th, No. 533 (submission on December 6th, 1999) in the commonly assigned patent, it is hereby incorporated by in full.

For all indications, can single dose or about 1 μ g/ days to about 5mg/ days dosage of divided dose or controlled, sustained release, or with every dose of about 0.01 μ g/kg to about 500 μ g/kg, more preferably from about 0.05 μ g/kg is to about 250 μ g/kg, most preferably be lower than about 50 μ g/kg, the administered peripherally novel cpd.Dosage can give 1,2,3 or 4 time in one day.Certainly, the dosage in these scopes changes with the effectiveness of every kind of analogue or derivative, and dosage can be determined by those skilled in the art.

In the methods of the invention, described polypeptide can (include but not limited to contain islet amyloid polypeptide or islet amyloid polypeptide analogue agonist to other compound and composition that reduction nutrition availability has long-acting or fugitive effect individually or with one or more, salmon calcitonin see calcimar or salmon calcitonin see calcimar agonist, cholecystokinin (CCK) or CCK agonist, Leptin (OB albumen) or Leptin agonist, Exendin or exendin peptide analogs agonist, other compound and the composition of GLP-1 or GLP-1 analogue agonist or PYY or PYY analogue or PYY related polypeptide) give together.Suitable islet amyloid polypeptide agonist for example comprises [25,28,29 Pro-] people's islet amyloid polypeptide (be also referred to as " tripro-amylin ", be set forth in United States Patent (USP) the 5th, 686,511 and 5,998, No. 367).The CCK that uses is preferably CCK octapeptide (CCK-8).Leptin for example is discussed in (Pelleymounter, Cullen etc., Science 269:540-543 (1995); Halaas, Gajiwala etc., Science 269:543-6 (1995); Campfield, Smith etc., Science 269:546-549 (1995)).Suitable Exendin comprises Exendin-3 and Exendin-4, and the Exendin agonist compound comprises those compounds that for example are described in PCT open WO 99/07404, WO 99/25727 and WO 99/25728.Suitable PYY polypeptide and analogue comprise and are described in U. S. application the: those compounds of [proxy's application case numbering 18528.740 and 18528.723].

Medicinal compositions

The invention still further relates to medicinal compositions, it comprises at least a The compounds of this invention or its pharmacologically acceptable salts of treatment or prevention significant quantity, together with pharmacy acceptable diluent, sanitas, solubilizing agent, emulsifying agent, auxiliary and/or be used to transmit the carrier of novel cpd.Such composition can comprise: the thinner of various buffer content things (for example acetate, Citrate trianion, tartrate, phosphoric acid salt, TRIS), pH and ionic strength; Additive, for example tensio-active agent and solubilizing agent (anhydrosorbitol monooleate ester for example, Yelkin TTS, Pluronics, Tween 20 and Tween 80, Polysorbate 20 and Polysorbate 80, propylene glycol, ethanol, PEG-40, sodium lauryl sulphate), antioxidant (monothioglycerol for example, xitix, acetylcysteine, sulphite (hydrosulphite and metabisulphite)), sanitas (phenol for example, meta-cresol, benzylalcohol, P-hydroxybenzoic acid (first, third, fourth) ester, benzalkonium chloride, trichloro-butyl alcohol, Thiomersalate, phenylmercuric salts (acetate, borate, and tonicity agents/raising agent (glycerine nitrate)), sodium-chlor, N.F,USP MANNITOL, sucrose, trehalose, glucose); Material is incorporated in the specific aggregation compound goods, for example poly(lactic acid), polyglycolic acid etc.; Or combine with liposome.Such composition will influence the interior rate of release of physical condition, stability, body and the interior clearance rate of body of The compounds of this invention.Referring to for example Remington ' sPharmaceutical Sciences 1435-712, the 18th edition, Mack Publishing Co., Easton, Pennsylvania (1990).

In general, in view of the pharmacological characteristics of The compounds of this invention, can the mode identical use The compounds of this invention with islet amyloid polypeptide.A preferable use is this novel cpd of administered peripherally, is used for the treatment of or prevents metabolic trouble and obstacle.Specifically, The compounds of this invention has the activity that conduct reduces nutrition availability, minimizing ingestion of food and influences the material of weight loss.

Compound of the present invention can be prepared and be used for administered peripherally, comprises that preparation is used to inject, gives in the oral administration, nose, other administration type of pulmonary administration, topical administration or those skilled in the art's approval.Examples of formulations is found in United States Patent (USP) 6,410, No. the 10/159th, 779, No. 511 and patent application, and it is hereby incorporated by in full.More particularly, can give medicinal compositions of the present invention, as long as can arrive target tissue through this approach by any common approaches.In a preferred embodiment, can be by any conventional periphery method, for example by in intravenously, intracutaneous, intramuscular, the breast, in the intraperitoneal, sheath, behind the eyeball, in the lung (for example terminal release); By oral, hypogloeeis, nose, anus, vagina or through the skin transmission, or, medicinal compositions is imported among the experimenter by at the privileged site operation transplantation.Treatment can be made up of single dose in for some time or multiple doses.Also imagined the controlled lasting release present composition.The example of microsphere technology is found in United States Patent (USP) the 6th, 458, No. the 5th, 578,708, No. 387 and United States Patent (USP), and it is hereby incorporated by in full.

Goods can be liquid, or can be solid, for example are used to the lyophilized solid of rebuilding.Waterborne compositions of the present invention comprises the novel cpd of significant quantity, its dissolving be dispersed in pharmaceutically acceptable carrier or aqueous medium in.Phrase " pharmacy or pharmacology are acceptable " refers to not produce the molecular entity and the composition of untoward reaction, transformation reactions or other inappropriate reaction when giving the animal or human." pharmaceutically acceptable carrier " used herein comprises any solvent, dispersion medium, Drug coating, antibacterial agent and anti-mycotic agent, isotonic agent and absorption delay agent etc.It is well-known in this area that such medium and material are used for the purposes of pharmaceutically active substances.Unless the medium of any routine or material and activeconstituents are incompatible, otherwise consider to use it for therapeutic composition.Also can join in the composition replenishing activeconstituents.In some cases, in single composition or solution, provide The compounds of this invention and the another kind of material (for example islet amyloid polypeptide, islet amyloid polypeptide agonist analogue, CCK or CCK agonist, Leptin or Leptin agonist, Exendin or exendin peptide agonists analogue or PYY or PYY analogue) of ingestion of food, reduction plasma glucose or change blood plasma lipide that reduces so that administration is very easily together.In other cases, other material and novel cpd are separated give may be more favourable.

The compounds of this invention can be mixed with tensio-active agent (for example hydroxypropylcellulose) suitably the aqueous solution of blended free alkali or pharmaceutically acceptable salt so that administration.Phrase used herein " pharmacologically acceptable salts " refers to the salt by the preparation of pharmacy acceptable (preferred nontoxic) bronsted lowry acids and bases bronsted lowry (comprising inorganic or organic bronsted lowry acids and bases bronsted lowry), include but not limited to the sulphur hydrochlorate, Citrate trianion, maleate, acetate, oxalate, hydrochloride, hydrobromate, hydriodate, nitrate, vitriol, hydrosulfate, phosphoric acid salt, superphosphate, Yi Yansuan salt, acetate, lactic acid salt, salicylate, Citrate trianion, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate salt, succinate, maleate, gentisate, fumarate, gluconate, saccharic acid salt, sugar lime, formate, benzoate, glutaminate, mesylate, esilate, benzene sulfonate, right-tosylate and pamoate (promptly 1,1 '-methylene radical-two-(2-hydroxyl-3-naphthoate)).Pharmacologically acceptable salts comprises those salt that form with free amine group, such as but not limited to deriving from hydrochloric acid, phosphoric acid, acetate, oxalic acid and tartaric those salt.Pharmacologically acceptable salts also comprises those salt that form with free carboxy, such as but not limited to those salt that derive from sodium hydroxide, potassium hydroxide, ammonium hydroxide, lithium hydroxide, calcium hydroxide, ironic hydroxide, Isopropylamine, triethylamine, 2-ethyl amido alcohol, Histidine and PROCAINE HCL, PHARMA GRADE.Under general storage and working conditions, these goods comprise sanitas, to prevent microorganism growth.

In one embodiment, prepare medicinal compositions of the present invention to such an extent that be suitable for parenteral and give, for example give by injection or infusion.Preferably, novel cpd is suspended in the aqueous carrier, for example in the isotonic buffer solution of pH about 3.0 to about 8.0, preferably at pH about 3.5 to about 7.4,3.5 to 6.0 or 3.5 to about 5.0 isotonic buffer solution.Useful buffer reagent comprises sodium acetate/acetate, Sodium.alpha.-hydroxypropionate/lactic acid, xitix, Trisodium Citrate-citric acid, sodium bicarbonate/carbonic acid, sodium succinate/succsinic acid, Histidine, Sodium Benzoate/phenylformic acid and sodium phosphate and three (methylol) aminomethane.Can use a kind of long-acting or " reservoir devices " sustained release preparation, in a plurality of hours or many days at percutaneous injection or after transmitting, the agent delivery of treatment significant quantity be gone in the blood flow.

The medicinal compositions that is suitable for injecting use comprises aseptic aqueous solution or dispersion liquid and is used for preparing immediately the sterile powder of aseptic injectable solution or dispersion liquid.With regard to all situations, formulation should be aseptic, and flowability should reach the degree that can be easy to inject.Also needing PPF polypeptide of the present invention is stable under production and condition of storage, and it must be anticorrosion, with the contamination of combating microorganisms (for example bacterium and fungi).Carrier can be solvent or dispersion medium, comprise for example water, ethanol, polyvalent alcohol (for example sorbyl alcohol, glycerine, propylene glycol and liquid macrogol etc.), N,N-DIMETHYLACETAMIDE, Cremorphor EL, its suitable mixture, and oil (for example soya-bean oil, sesame oil, Viscotrol C, Oleum Gossypii semen, ethyl oleate, Isopropyl myristate, Tetrahydrofurfuryl polyethylene glycol ether (glycofurol), Semen Maydis oil).For example by using Drug coating (for example Yelkin TTS), under the dispersion liquid situation, pass through to keep required granular size and, can keeping suitable flowability by using tensio-active agent.Can utilize various antibacterial agents and anti-mycotic agent, for example meta-cresol, benzylalcohol, P-hydroxybenzoic acid (first, the third, fourth) ester, trichloro-butyl alcohol, phenol, phenylmercuric salts (acetate, borate, nitrate), Sorbic Acid, Thiomersalate etc. reach the prevention to microbial process.In many cases, preferably comprise isotonic agent (for example sugar, sodium-chlor).By in composition, using absorption delay agent (for example monostearate aluminium and gelatin), can realize that the absorption of injectable composition prolongs.Representational medicinal compositions can be the water system solution of 0.1-5% The compounds of this invention, be buffered to about 3.0 to about 6.0 about 0.02% acetate, phosphoric acid salt, Citrate trianion or glutaminate together with pH to about 0.5% (w/v) with final composition, and the carbohydrate of about 1.0-10% (w/v) or polyvalent alcohol tonicity agents; With the sanitas of the about 0.005-1.0% (w/v) that chooses wantonly, it is selected from meta-cresol, benzylalcohol, p-Hydroxybenzoate and phenol.

Can be by being added in the active compound of the aequum in the suitable solvent, and various other compositions of enumerating more than adding as required, then Sterile Filtration, preparation aseptic injectable solution.In general, by various activeconstituentss through sterilization are joined in the aseptic solvent, prepare dispersion liquid, wherein aseptic solvent contains basic dispersion medium and above required other composition of enumerating in the material.For the sterile powder that is used to prepare aseptic injectable solution, preferred manufacturing procedure is vacuum-drying and the Freeze Drying Technique that is added the powder of any other required composition by the solution generation activeconstituents of its previous Sterile Filtration.

In general, determine the treatment of novel cpd of the present invention or prevent significant quantity according to age, body weight and receptor's the physical appearance or the seriousness of disease or metabolic disorder or obstacle.Referring to for example Remington ' s Pharmaceutical Sciences 697-773.In addition referring to Wang and Hanson, Parenteral Formulations of Proteins and Peptides:Stability and Stabilizers, Journal of Parenteral Science and Technology, Technical Report No.10, Suppl 42:2S (1988).Usually, can use about 0.001 μ g/kg body weight/day, but recognize, can use more or fewer dosage as skilled practitioners to the dosage of about 1000 μ g/kg body weight/day.Administration can be once a day or repeatedly, or frequency is lower, can combine with other composition described herein to give.Should be pointed out that and the invention is not restricted to the dosage that this paper mentions.

Experiment that can be by using the mensuration metabolic trouble established or obstacle level is determined proper dosage together with relevant dose-response data.Final dosage regimen is considered to change pharmaceutically-active factor by the attending doctor and is determined, these factors for example are seriousness, administration time and other clinical factor of the ratio work of medicine, the seriousness of damage and patient's responsiveness, patient's age, physical appearance, body weight, sex and meals, any infection.Along with the carrying out of research, will manifest about the suitable dose level of specified disease and illness and the more information of treatment time-histories.

The effective dose that gives with single dose or divided dose was generally about 1-30 μ g to about 5mg/ days, and preferably about 10-30 μ g was to about 2mg/ days, and more preferably from about 5-100 μ g was to about 1mg/ days, and most preferably from about 5 μ g were to about 500 μ g/ days.Dosage can be about 0.01 to about 500 μ g/ agent.Imagining compound of the present invention can give 1,2,3,4 time or more times on the 1st.Therefore, representative dosage can come from the administration total amount of every day and the administration number of times of every day.For example, representative type dosage can be about 0.125 μ g/ agent (give 0.5 μ g every day 4 times) to about 5mg/ agent (giving 5mg every day one time).Other dosage can be about 0.01 to about 100 μ g/kg/ agent.Representative dosage in addition can be 20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190 or 200 μ g/ agent.The definite dosage that gives can determine that it depends on the effectiveness of particular compound by those skilled in the art, and individual age, body weight and physical appearance.Give and when for example needing to suppress the nutrition availability, reducing ingestion of food, body weight, blood sugar or blood plasma lipide, to begin, for example, when illness has sign at first or after diagnosing out obesity, diabetes or insuline resistance syndrome, begin soon.Can give for example injection (preferred subcutaneous or intramuscularly), oral, nose, through skin etc. by any approach.The dosage that is used for some approach (for example orally give) can increase, and for example increases about 5-100 doubly, thereby causes that bioavailability reduces.

In one embodiment, when wanting parenteral to give medicinal preparations, compositions formulated, so that transmit the novel cpd dosage of 1 μ g/kg to 100mg/kg body weight/day, preferred 10 μ g/kg are to the dosage of about 50mg/kg body weight/day.Can week initial heavy dose carry out parenteral and give, then continue infusion, to keep the treatment cyclical level of medicine.Persons skilled in the art are easy to optimize effective dose and dosage regimen, and this can determine according to the clinical disease of good medical practice and individual patient.

Administration frequency depends on the pharmacokinetic parameter and the route of administration of medicine.Those skilled in the art determine best pharmaceutical preparation according to route of administration and required dosage.Referring to for example Remington ' s Pharmaceutical Sciences, ibid, the 1435-1712 page or leaf.Such preparation can influence the interior rate of release of physical condition, stability, body and the interior clearance rate of body of the medicine that gives.According to route of administration, can calculate proper dosage according to body weight, body surface area or organ size.Persons skilled in the art need not improper undo experimentation, can determine the necessary meticulousr calculating of appropriate therapeutic dosage in a usual manner, especially according to dosage information disclosed herein and experiment and in the clinical experiment of animal or human's body under the situation of observed pharmacokinetic data.

Recognize that medicinal compositions of the present invention and methods of treatment can be used for human medicine and veterinary applications.Therefore, the experimenter that treat can be Mammals, preferably is people or other animal.For purpose for animals, the experimenter comprises for example domestic animal (comprising cow, sheep, pig, horse and goat), pet (for example dog and cat), rare (exotic) animal and/or zoo animal, laboratory animal (comprising mouse, rat, rabbit, cavy and hamster); And poultry, for example chicken, turkey, duck and goose.

For helping to understand the present invention, include following embodiment in.Relating to embodiments of the invention should not be interpreted as certainly to concrete restriction of the present invention; these of limit of power that now known or later exploitation of the present invention belongs to those skilled in the art change, and are considered to fall into the scope of the invention described herein and hereinafter claimed.

Embodiment

Embodiment 1: polypeptide synthetic that reduces calorie intake

Can use the standard polypeptide synthesis method to synthesize following polypeptide.Such method is described in hereinafter and United States Patent (USP) 6,610, and 824 and United States Patent (USP) 5,686,411 and patent application serial number the 454th, No. 533 (submission on December 6th, 1999), these documents are hereby incorporated by in full.

Go up amino acid (Applied Biosystems, Inc.) the assembling polypeptide that uses the Fmoc-protection at 4-(2 '-4 '-Dimethoxyphenyl)-Fmoc amino methyl phenoxy-acetamide nor-leucine mbha resin (Novabiochem, 0.55 mmole/g).In general, in whole synthesizing, use single coupling circulation, and use Fast Moc (HBTU activation) chemistry.But, may be lower in some position link coupled efficient than expection, need double couple crosslinking.Use piperidines to go the protection (removals of Fmoc group) equally may be always not effective, need two going to protect the peptide chain that increases.Use triethyl silicane (0.2mL), dithioglycol (0.2mL), methyl-phenoxide (0.2mL), water (0.2mL) and trifluoroacetic acid (15mL) mixture; according to standard method (Introduction to CleavageTechniques; Applied Biosystems; that Inc.), realizes having finished peptide finally goes protection.Peptide precipitates in ether/water (50mL) and is centrifugal.Be deposited in the glacial acetic acid and rebuild, and freeze-drying.Freeze dried peptide is dissolved in the water.Measure crude product purity then.

In purifying and analytical procedure, use solvent orange 2 A (aqueous solution of 0.1%TFA) and solvent B (the ACN solution of 0.1%TFA).

To contain preparation type C-18 post and purifying (in 40 minutes, containing the solvent orange 2 A solution of 10%-40% solvent B) on the solution of each peptide species.Use the purity of degree mensuration separation fractions such as C-18 analysis mode post.Merge pure separation fraction so that above discriminating peptide to be provided.The freeze-drying peptide is carried out analysis mode RP-HPLC (gradient be 30 minutes include the solvent orange 2 A solution of 30%-60% solvent B), to measure retention time.

Embodiment 2: the receptors bind experiment

At first, polypeptide can be used to measure the binding ability with islet amyloid polypeptide, thyrocalcitonin and CGRP acceptor in experiment.Measure to combine with islet amyloid polypeptide acceptor, Calcitonin Receptor and CGRP acceptor interactional to test and for example be described in United States Patent (USP) the 5th, 264, No. 372, it is hereby incorporated by in full.

More particularly, following The compounds of this invention is estimated with combining of islet amyloid polypeptide acceptor.(Arlington Heights IU.) buys by Amersham Corporation ¹²⁵I-rat Langerhans islet amyloid polypeptide (Bolton-Hunter is marked at N end Methionin).Unlabelled peptide derive from BACHEM Inc. (Torrance, Calif.) and Peninsula Laboratories (Belmont, Calif.).

The male Sprague-Dawley_ rat of sacrificed by decapitation (200-250g).Take out brain, and place cold phosphate-buffered saline (PBS).From the outside of belly, to hypothalamic otch, the side is the boundary with the tractus olfactorius, and is extended with 45 to the midline by these tractus olfactorius by mouth in making.This basal forebrain tissue comprises nucleus accumbens septi and peripheral region, to its weigh and ice-cold 20mM HEPES damping fluid (20mM HEPES acid, in 23 ℃ with NaOH with pH regulator to 7.4) in homogenate.By centrifugal 15 minutes, clean film 3 times with fresh buffer with 48,000 * g.With final film pellet resuspended in containing the 20mM HEPES damping fluid of 0.2mM phenylmethylsulfonyl fluoride (PMSF).

For detecting ¹²⁵The combination of I-islet amyloid polypeptide will derive from the film and the 12-16pM of 4mg original wet re-organized ¹²⁵The 20mM HEPES buffered soln of I-islet amyloid polypeptide (it contains 0.5mg/ml bacitracin, 0.5mg/ml bovine serum albumin and 0.2mM PMSF) incubation.Solution was in 2 ℃ of incubations 60 minutes.(N.J.) filtration stops incubation for Whatman Inc., Clifton, and this filter soaked 4 hours in 0.3% polymine in advance, so that reduce the non-specific binding of radiolabeled peptides by the GF/B glass fibre filter.Face and filter the cold PBS cleaning filter of the preceding 5ml of using, after filtration, use the cold PBS cleaning filter of 15ml immediately.Take off filter, the counting efficiency with 77% in γ-counter is evaluated and tested radioactivity.By 10 ^-12-10 ^-6There is detection combination down in the unmarked test compounds of M, the curve of competing, and by using non-linear regression (the Inplot program of 4-parameter logistic equation; GraphPAD Software, San Diego) the analysis competition curve.

In this experiment, people's islet amyloid polypeptide of purifying is with the IC of about 50pM of detection ₅₀In conjunction with its acceptor.Test compounds of the present invention the results are shown in following table, show that every kind of compound all has significant receptor-binding activity.

Basic as to the description evaluation The compounds of this invention of islet amyloid polypeptide and combining of CGRP acceptor, only be to use ¹²⁵I-hCGRP and by the film of SK-N-MC cell preparation, and known SK-N-MC cell expressing CGRP acceptor (Muff, R. etc., Ann NY Acad.Sci.1992:657,106-16).As the description of islet amyloid polypeptide being carried out the combination experiment, only be to use 13,500cpm ¹²⁵I-hCGRP/ hole or 21.7 pM/ holes (Amersham).

As known in the art, can use the Chinese hamster ovary celI of same expression Calcitonin Receptor or T47D cell Muff R. etc., Ann N YAcad Sci.1992,657:106-16 and KuestnerR.E. etc., Mol Pharmacol.1 994,46:246-55) research and the combining of Calcitonin Receptor.

Table X. the EC of polypeptide ₅₀Value (nM)

Compound	Islet amyloid polypeptide	Thyrocalcitonin	CGRP
					1	0.026	0.029	2.342
2	0.047	0.052	33.988
				3	0.023	0.020	0.490
4	0.035	0.019	8.500
				5	0.022	0.018	2.600
6	0.030	nt	nt
				7	0.057	nt	7.540
8	8.070	0.478	175.665
				9	0.043	0.014	1.600

Not test of nt representative.

Embodiment 3: polypeptide is to the activity of ingestion of food

With the little time of 12:12: dark cycle, grouping stable breeding female NIH/Swiss mouse (8-14 age in week).Water and standard ball shape mouse feed can arbitrarily obtain, and indicate except the place.Animal begins fasting when preceding 1 day about 15:00 of experiment.

During in time=0 minute, give all animal peritoneal injection solvent or polypeptide with the amounts of 200 μ l/ mouse, and the standard feed of give immediately to weigh in advance (10-15g).Took out foods in 30,60,120 and 180 minutes and weigh, to determine food consumption quantity.Treatment is expressed as with respect to the variation percentage that contrasts the effect of ingestion of food.

As seen from Figure 1, the compound 2 of 25-300nmol/kg dosage reduces ingestion of food on back 30 minutes dose-dependently ground of injection.The ingestion of food that following table has been described with 25nmol/kg dosage administered peripherally (peritoneal injection) polypeptide reduces.The decline percentage of the data represented accumulation ingestion of food with respect to solvent of time point 30,60,120 and 180 minutes.

Compound	30 minutes	60 minutes	120 minutes	180 minutes
					1	-58	-46	-33	-22
2	-58	-54	-52	nt
					3	-58	-52	-37	-33
4	-42	-31	-35	-30
					5	-66	-53	-29	-27
6	-48	-45	-23	nt
					7	-60	-52	-23	nt
8	-6	-15	-25	-28
					9	-80	-64	-43	nt

10	-19	-20	-35	nt
					11	-52	-47	-38	-35
12	-43	-39	-37	-32
					13	-40	-33	-25	-24
14	-52	-36	-28	-33
					15	-67	-59	-37	-30
16	-26	-29	-30	-27
					17	-42	-30	-30	-25
18	-2	-7	-16	-21
					19	-25	-25	-35	-31
20	-9	-21	-30	-31
					21	9	-5	-18	-18
22	-11	-20	-31	-30
					23	8	0	-19	-12
24	-40	-34	-35	-35
					25	-29	-34	-45	nt
26	-29	-36	-47	nt
					27	-12	-11	-32	nt
28	-8	-16	-28	nt
					29	4	-1	-25	nt
30	-1	-2	-19	nt
					31	-11	-18	-23	nt
32	-15	-21	-31	nt
					33	-7	-10	-15	nt
34	-11	-6	-16	nt

35	-20	-16	-18	nt
					36	-34	-22	-24	-25
37	-3	-2	-16	nt
					38	-24	-13	-8	nt
39	7	-14	-23	nt
					40	-11	-5	-2	nt
41	-4	-9	-12	nt
					42	-11	-18	-32	nt
43	-4	-7	-18	nt
					44	-6	-13	-25	nt
45	-13	-7	-3	nt
					46	-6	-11	-16	nt
47	-5	-13	-27	nt
					48	-54	-51	-36	nt
49	-33	-26	-25	nt
					50	-70	-62	-48	nt
51	-44	-39	-35	nt
					52	-29	-24	-23	nt
53	-92	-89	-36	nt
					54	1	-4	-10	nt
55	9	-5	-12	nt
					56	4	-13	-16	nt
57	-18	-24	-23	nt
					58	-62	-51	-29	nt
59	-81	-77	-50	nt

60	-43	-40	-26	nt
					61	-23	-27	-32	nt
62	-14	-22	-38	nt
					63	-19	-22	-28	nt
64	-65	-58	-44	nt
					65	-33	-29	-32	nt
66	-13	-15	-28	nt
					67	-10	-11	-12	nt
68	-10	-13	-21	nt
					69	-29	-31	-45	nt
70	-76	-64	-47	nt
					71	-7	-13	-22	-18
72	0	-8	-13	-19
					73	-51	-31	-23	-28
74	-42	-32	-31	nt
					75	-60	-52	-38	nt
76	-25	-29	-40	nt
					77	-46	-43	-44	nt
78	-57	-44	-44	nt
					79	-49	-40	-33	nt
80	-32	-28	-22	nt
					81	-28	-24	-33	nt
82	-7	-13	-16	-19
					83	-7	-13	-22	-12
84	-53	-40	-20	nt

85	3	-16	-16	nt
					86	-44	-26	-16	nt
87	-43	-32	-21	nt
					88	-64	-61	-39	-22
89	-6	-13	-22	-20
					90	-55	-41	-24	-15
91	-59	-47	-26	-24
					92	-31	-29	-30	-27
93	-43	-30	-27	-29
					94	-62	-42	-36	-31
95	-81	-69	-34	-31
					96	-49	-38	-19	-23
97	-78	-76	-60	-40
					98	-18	-13	-5	-1
99	-57	-55	-50	nt
					100	-60	-52	-41	nt
101	-52	-48	-35	nt
					102	-58	-53	-45	nt
103	-50	-44	-30	nt
					104	-69	-67	-54	nt
105	-83	-82	-52	nt
					106	-58	-54	-39	nt
107	-84	-78	-47	nt
					108	-70	-66	-38	nt
109	-61	-54	-43	nt

110	-80	-72	-59	nt
					111	-39	-37	-32	nt
112	-62	-65	-50	nt
					113	-79	-86	-55	nt
114	-17	-20	-25	nt
					115	6	-3	-25	-25
116	5	5	3	nt
					117	-13	-11	-3	nt
118	-4	0	13	nt
					119	6	-8	-11	nt
120	-3	1	-6	-7
					121	5	2	-1	3
122	-6	-12	-23	-21
					123	1	-13	-17	-13
124	4	-4	-15	-16
					125	10	-1	-6	nt
126	5	-10	-20	nt
					127	-5	-14	-12	-12

Nd=does not implement.

Embodiment 4: The compounds of this invention is to losing weight and the activity of calorie intake

Make the male Sprague-Dawley_ rat (350g of independent stable breeding; 12 little time/dark cycle) keep high fat diet (58% kilocalorie is from fat) 4 weeks.When the getting fat phase finishes, implanting 14 days osmotic pump (Durect Corp.) under the anesthesia between omoplate.Rat is accepted the solvent (50%DMSO) of pump sustained delivery or the polypeptide of 2.9nmol/kg/ days dosage.Obtain ingestion of food and body weight detected result weekly.Fig. 2 A and 2B have shown that calorie intake decline and weight increase that polypeptide compound 3, compound 4 or compound 5 produced descend in whole 14 day testing period.

Following table provides the weight loss percentage of several compounds in the 1st week and the 2nd week.

Table X gives the weight loss after the representative compounds of the present invention

Compound	The 1st week	The 2nd week
			Compound
1	8.3 *	10.5 *
			Compound 2	9.8 *	9.4 *
Compound 3	5.9 *	6.7 *
			Compound 4	6.8 *	9.2 *
Compound 5	8.6 *	11.3 *
			Compound 6	2.9 *	3.8 *
Compound 7	10.0 *	11.4 *
			Compound 8	2.3	2.5
Compound 9	4.9 *	4.9 *

^*Compare P＜0.05 with contrast

Embodiment 5: organization

Make the male Sprague-Dawley_ rat (420g of independent stable breeding; 12 little time/dark cycle) keep high fat diet (58% kilocalorie is from fat) 4 weeks.When the getting fat phase finishes, implanting 14 days osmotic pump (Durect Corp.) under the anesthesia between omoplate.Rat is accepted the solvent (50%DMSO) of pump sustained delivery or the compound 1 of 70nmol/kg/ days dosage.Put to death animal at the 12nd day.Freezing immediately corpse, and by chemical analysis (Covance Laboratories, Madison, WI) measurement organization (fat and albumen).Fig. 3 shows that than contrast, the percentage that the lipid content that compound 1 is handled rat accounts for exchange physique amount descends.In addition, compound 1 increases the percentage of lean tissue mass's content.

Embodiment 6: stomach emptying and ionized calcium

By measuring the appearance monitoring stomach emptying of tube feed tritiate glucose in the blood plasma.The experimenter is sentient male Sprague Dawley_ rat (7-9 week age, 12:12 hour light dark period), and it can arbitrarily obtain food and water, until the experiment beginning.Before administration, withdraw food and water.In the time of t=-5 minute, give peptide or solvent (200 μ l salt solution) simultaneously.In the time of t=0 minute, the through port pharyngeal canal gives 1ml and contains 5 μ Ci D-[3-3H] glucose (Dupont, Wilmington, DE, aseptic aqueous solution USA).In the time of t=20 minute, the tail point is used toponarcosis (Hurricaine_, 20% Benzocaine liquid).In the time of t=40 minute,, collect about 250 μ l blood to the heparinization pipe with scalper ligation tail point.(Medfield MA) detects the ionised calcium of blood plasma immediately for Ciba/Corning, Inc. to use Ciba/Corning 634Ca/pH analyser then.With pipette, extract 10 μ l plasma samples to the flicker bottle of preparation (in the 0.5ml water+2ml flicker mixture (Ecolite scintillation cocktail ICN, Costa Mesa, CA)), vortex mixed, and at beta-counter (1209 Rack-beta; LKB-Wallac, Gaithersburg, MD) in 1 minute/bottle counting.

In Fig. 4 A, point is represented the mean value ± standard deviation of 6 SD rats (feed and raise, consciousness is arranged).The peptide of dosage shown in the subcutaneous injection when t=0.Collect blood after 35 minutes and carry out the cpm analysis. ^*Have a few all is p＜0.001 to saline control; ANOVA, Dunnett check.According to non-linear regression: ED50=2.3 μ g/kg.Bottom=25cpm/10 μ l; Top=328cpm/10 μ l.The maximum of blood plasma cpm is reduced to :-92%.The goodness of fit: r2=0.9992.

In Fig. 4 B, point is represented the mean value ± standard deviation of 6 SD rats (feed and raise, consciousness is arranged).The peptide of dosage shown in the subcutaneous injection when t=0.Collect blood after 35 minutes and carry out the cpm analysis. ^*Relative saline control P＜0.001; ANOVA, Dunnett check.According to non-linear regression: ED50=1.1 μ tg/kg.Bottom=1.2mmol/L; Top=1.3mmo1/L.The maximum of blood plasma iCa is reduced to :-14%.The goodness of fit: r2=0.9936.

Embodiment 7: triglyceride level

In research 1, feed and raise kind of a retired female Harlan Sprague-Dawley_ rat (HSD; HarlanTeklad, Indianapolis, IN) (40% calorie is from fat for high fat diet; Feed code T D95217 HarlanTeklad) reached for 9 weeks, began one's study then, kept this feed in whole research cycle.In whole research, one group of rat arbitrarily feeds to be raised, and handles (n=10) with respect to the metabolism that contrasts-be untreated rat (n=10) to check representative compounds-compound 1 of the present invention.For measuring the metabolism of compound 1 in setting up food restriction (" fixed food "), give 75% of the preceding baseline ingestion of food of its research of second group of rat every day and reach 10 days, begin compound 1 (n=10) or solvent (n=10) then and handle (with respect to body weight before the restriction, weight loss about 5%).At this moment, all rats all be received under the isoflurane anesthesia Alzet_ osmotic pump that subcutaneous operation is implanted in the territory, interscapular region (Durect, Cupertino, CA).Pump prepares to be used for to continue to transmit in 21 days compound 1 (101 μ g/kg/ days) or solvent (aqueous solution of 50% dimethoxy sulfoxide [DMSO]).The limited rat of food continues to give 75% of its baseline ingestion of food every day.After handling in 21 days, by cardiac puncture by the absorption of isoflurane anesthesia after rat (at the about 2-4 of fasting in the morning hour) blood sample is obtained to the heparinization syringe, the blood plasma of acquisition is used for analyte analyzation.Use is used for standard automated analysis (LabCorp, Inc., San Diego, CA) the mensuration plasma triglyceride of clinical chemistry.

In research 2, ((32% calorie is from fat for male rat high fat diet MA) for CharlesRiver, Wilmington to feed the HSD rat Levin strain of raising lipophilia; Feed numbering 12266B, Research Diets, Inc., New Brunswick NJ) reached for 6 weeks, begins one's study then, and keep this feed in whole experiment.Place the subcutaneous osmotic pump that contains compound 1 or DMSO solvent according to research 1, begin to handle from this moment, and continue 3 weeks (21 days).Comparative compound 1 was handled rat (303 μ g/kg/ days; N=10), vehicle treated contrast (n=10) and accept every day compound 1 handle rat eaten food amount coupling hello raise metabolizing parameters between the group (n=10).Carry out blood according to research 1 and collect and the plasma triglyceride concentration analysis, but use the tail vein to collect in the 1st week and the 2nd time-of-week point.

Study 3 according to research 2, just handle 8 weeks of animal (56 days) with the compound 1 of various dose.At the 28th day that handles, used pump with fresh osmotic pump replacement.Compound 1 (n=10/ group) or DMSO solvent that pump is used to transmit 1,3,10 or 100 μ g/kg/ days dosage (contrast, n=10).Pairing is in addition fed and to be raised group (n=10) and accept solvent, and every day hello raise 1 group of abundance of food of being eaten of 100 μ g/kg/ days compounds.Be collected in the heparinization pipe by the tail vein, raise the plasma triglyceride concentration that rat is collected blood and analyzes baseline, 2,4 and 6 time-of-week points by there being consciousness to feed; Raise rat by cardiac puncture by feeding of isoflurane anesthesia and collect blood in the heparinization syringe, use standard triglyceride level experiment (Cobas Mira Plus, Roche Diagnostics) to analyze the plasma triglyceride concentration of the 8th time-of-week point.

With the one-factor analysis of variance (ANOVA) succeeded by Dunnett multiple comparisons check (Prism V.4.01, GraphPad Software, San Diego, CA) in the evaluation study preset time point handle between the difference of average blood plasma triglyceride levels.Statistically, p＜0.05 o'clock thinks that difference is significant.

As be respectively shown in research Fig. 5-7E of 1,2 and 3, cause substantial reduction in triglycerides concentration with compound 1 processing at 1-8 in week.Fig. 5 has shown at usefulness compound 1 plasma triglyceride concentration in the retired rat of female breeding after continuing subcutaneous (s.c.) infusion to handle 21 days.With arbitrarily feed the contrast of raising and compare, the rat that gives compound 1 shows significantly reduced triglyceride levels: no matter be to give compound 1 under the condition arbitrarily feeding to raise, still give compound 1 under the food restricted condition, all observe this effect.With arbitrarily feed the vehicle treated contrast of raising and compare, ^*P＜0.05, ^*P＜0.01.

Fig. 6 A-6C has described in by 3 weeks that continue 1 processing of h inf compound, the plasma triglyceride concentration in the male Levin rat of meals inducing obesity (DIO).Figure representative is by handling (A) 1 week, (B) 2 weeks and (C) value that blood sample obtained during 3 weeks.Compare with the contrast of vehicle treated, ^*P＜0.05, ^*P＜0.01.

Fig. 7 A-7E has shown the dose response research of compound 1, compares with the solvent contrast of 8 time-of-week points in it with 2,4,6, and the dosage that was low to moderate 1 μ g/kg/ days causes plasma triglyceride concentration decline (being respectively Fig. 7 B, C, D and E).Compare with the contrast of vehicle treated, ^*P＜0.05, ^*P＜0.01.Treatment group is with pictorial representation.

Embodiment 8:ghrelin experiment

This embodiment provides and has detected the representativeness experiment of The compounds of this invention to the ghrelin effect.

With the little time of 12:12: dark week is aspire to 22.8 ± 0.8 ℃ of male Harlan SpragueDawley_ of stable breeding (HSD) rat.All experiment is all carried out in the photoperiod.About 20 hours of animal fasting before experiment.All animals all can freely obtain water, until the experiment beginning.With 20% Benzocaine (Hurricaine, Beutlich Pharmaceutical, Waukegan, IL) anesthetized animal tail is collected blood sample by the tail vein.Use Linco RIA test kit GHRA-89HK and GHRA-88HK to detect total ghrelin concentration and active ghrelin concentration respectively.

In research 1, by the tail of toponarcosis the HSD rat is carried out regular blood sampling, and detect the ghrelin level.When t=0, to rat (n=6) subcutaneous injection 125 μ g/kg pentagastrins (Sigma), with gastric acid secretion (in Fig. 1 PG=0 minute), subcutaneous (s.c.) injection 10 μ g rat Langerhans islet amyloid polypeptides after 20 minutes.Analyze the total ghrelin and activity (acidylate) ghrelin (Linco) of blood sample.Shown in Fig. 8 A, islet amyloid polypeptide reduced active ghrelin about 50% in 1 hour.

Study 2, whether being independent of pentagastrin with check external source islet amyloid polypeptide stimulates and suppresses the ghrelin secretion.Give fasting rat skin lower injection salt solution or 30 μ g/kg rat Langerhans islet amyloid polypeptides during in time=0 minute, or subcutaneous injection salt solution or 125 μ g/kg pentagastrins (Sigma, lot number 050K1525).When being 20 minutes, gives the time rat Langerhans islet amyloid polypeptide (AC0128 of 30 μ g/kg dosage in 100 μ l salt solution by subcutaneous injection, lot number AR2081-42A, Amylin Pharmaceuticals) or independent salt solution solvent (being respectively n=5,5).At least collect plasma sample at 0,10,20,30,60 and 90 minute time point.Fig. 8 B and 8C all show, compare with saline control, give islet amyloid polypeptide total blood plasma ghrelin is descended, and Fig. 8 B confirms: compare with contrast, blood plasma ghrelin descends when having (promptly not having pentagastrin) at independent islet amyloid polypeptide.As if pentagastrin strengthen the ghrelin reduction effect of islet amyloid polypeptide.

Embodiment 9: pancreas function

In the HSD of fasting, anesthesia rat (heavy 320-350g), carry out femoral artery and femoral vein cannulation, and it was stablized 90 minutes.When t=-30, beginning infusion of saline (1ml/ hour) or compound 1 (1ml/ hour).When t=0, rat is accepted imp and injects 10 μ g/kg caerulin.Different time points in t=-30 to t=240 is collected blood plasma amylase, lipase sample.

Shown in Fig. 9 A and 9B, weakened the active increase of pancreatin in the acute pancreatitis in rats model blood with compound 1 processing, this prompting islet amyloid polypeptide agonist may be prevention and treat the pancreatitic prospect candidate medicine that has.

Embodiment 10: the gastric acid secretion of islet amyloid polypeptide

With the little time of 12:12: dark week is aspire to 22.8 ± 0.8 ℃ of male Harlan SpragueDawley_ of stable breeding rats.Experiment was carried out in the photoperiod.Feed and raise rat feed (Teklad LM 485, Madison, about 20 hours of the fasting before experiment of rat WI).They can freely obtain water, until the experiment beginning.

Underwent operative makes two gastric fistula pipes of rat (9-14 week age, body weight 264-395g) equipment supply merchant (Zivic Miller, catalog number (Cat.No.) SCAO3.00).In the laparotomy process, under halothane anesthesia, annular two-chamber plug is sewn in the coat of the stomach.Two 2.3mm internal diameter silicon rubber (advance and go out) are telescopic joint to communicate to plug and with gastral cavity, and by stomach wall these two sleeve pipes to the interscapular region, wherein these two sleeve pipes expose at stomach wall and interscapular region respectively subcutaneous in tracking.With clamp sealing laparotomy wound, rat was positioned in the recovery cage of heating 1 day, can freely obtain water.After this, map is brought mouse up separately, can freely obtain water and rat feed, stands overnight fast until about 10-15 angel rat after operation, is used for the experiment to there being the consciousness rat to carry out.

Open stomach catheter, and be connected to flexible PE240 pipe, be used for injection and sampling.For guaranteeing catheter patency, inject the salt brine solution of 2-3mL room temperature, and immediately by extracting out in the stomach.Repeat this step, until being easy to mobile and the effluent clarification., through another conduit it is extracted out immediately then by injection 5mL salt solution and 2mL air through a conduit, detect gastric acid secretion with 10 minutes intervals.Like this, the error that produces owing to the little secretion volume of incomplete extraction can be reduced to greatest extent.Use pH meter (Beckman model number PHI34Fullerton, CA), with 0.01N sodium hydroxide with each stomach extract titration of 3mL to pH7.0.Use the needed alkali number of each titration (proofreading and correct according to extracting cumulative volume out) to calculate the sour mole number of each sample.

After collecting baseline sample and writing down recovery volume, give animal subcutaneous injection 125 μ g/kg pentagastrins (Peninsula Laboratories lot number 019945 and 034686), and stomach sampling in 10 minutes is continued 2 hours again.Injected back 40 minutes at pentagastrin, observe gastric acid secretion constantly in this and reach stable platform, rat Langerhans islet amyloid polypeptide (lot number AR905-80 with salt solution (n=6) or 0.01,0.1,1.0,10.0 or 100 μ g (being respectively n=3,3,4,5,5) dosage, Amylin Pharmaceuticals Inc., San Diego, CA) subcutaneous injection rat.

Shown in Figure 10 A, the pentagastrin gastric acid secretion reaches 4.6 times, injects back 40 minutes about 63.4 ± 3.3 μ mol/10 minute (population means by about 13.8 ± 2.2 μ mol/10 minutes baseline rate to pentagastrin; P＜0.0001).40 minutes injection islet amyloid polypeptide dose-dependently ground gastric acid inhibitories produce behind pentagastrin, and the half life that 0.1,1,10 and 100 μ g dosage play work week was respectively 8.6,10.4,5.8 and 6.3 minutes.When the highest (100 μ g) dosage islet amyloid polypeptide, the gastric acid secretion of pentagastrin stimulation in 1 hour reduces by 93.4 ± 2.6% (for dosage 0.1-100 μ g, P＜0.001) behind the injection islet amyloid polypeptide.This secreting rate is only injected 32% (P＜0.01, t check, Welch correction) of preceding baseline rate for pentagastrin.The dose response that islet amyloid polypeptide suppresses the gastric acid secretion of pentagastrin stimulation is shown in Figure 10 B.The inhibiting ED of the hydrochloric acid in gastric juice of islet amyloid polypeptide ₅₀It is 0.05 μ g/ rat ± 0.15 log unit (41pmol/kg).

Embodiment 11: the stomach provide protection of islet amyloid polypeptide

In tube feed 1mL dehydrated alcohol (ethanol-200 standard dewatered ethanol, USP, SpectrumQuality Products, Inc.Gardena, CA) preceding 20 minutes, with 0.1mL salt solution (n=12) or with the fasting male rat of equal volume (being respectively n=5,5,5,9,9,5,6) the subcutaneous injection body weight 163-196g that contains 0.001,0.01,0.1,0.3,1,3 or 10 μ g dosage rat Langerhans islet amyloid polypeptides.Behind tube feed 30 minutes, with every rat of 5% halothane anesthesia, the excision stomach was opened along lesser gastric curvature, and is turned up, to expose mucous membrane.With the stomach that the drip washing of light and slow ground of salt solution is turned up, each people who handles blind viewer by 10 experiments grades to mucosa injury immediately.The evaluation dimensional constraints is not between 0 (observing damage) and 5 (mucomembranous surface 100% is covered by hyperemia, ulcer or slough formation), the 0-5 score system that uses with other people (for example Guidobono F. etc., Br JPharmacol 120:581-586 (1997)) is suitable.Figure 11 A has shown the damage of representing with the percentage of the alcohol induced damage result that keeps the score.

For whether the stomach provide protection of measuring islet amyloid polypeptide is attributable to the islet amyloid polypeptide specific mechanism; preceding 25 minutes of ethanol tube feed to 4 rat intravenous injection 3.0mg selectivity islet amyloid polypeptide antagonist AC187 (Amylin Pharmaceuticals; Inc.San Diego; CA), (is t=-20 minute apart from tube feed) subcutaneous injection 0.3 μ g rat Langerhans islet amyloid polypeptide after 5 minutes.The excision stomach, and as mentioned above behind the ethanol tube feed 30 minutes to damage grading.The result is provided in the penult bar rod among Figure 11 A.Last bar rod has shown the result who injects 3.0mg AC187 and do not inject the rat Langerhans islet amyloid polypeptide.Figure 11 B has shown the result has more selectively been investigated.

Figure 11 C shows, avoids mucosa injury (for the dosage of 0.1 μ g and Geng Gao, P＜0.05) at the islet amyloid polypeptide dose-dependently ground protection stomach that the subcutaneous injection in preceding 5 minutes of ethanol tube feed gives.0.3 μ g and more the islet amyloid polypeptide of high dosage damage kept the score to reach reduce by 67%.The ED of islet amyloid polypeptide stomach provide protection in this experimental system ₅₀It is 0.036 μ g (31pmol/kg) ± 0.40 log unit.

For whether the stomach provide protection of further studying islet amyloid polypeptide can take place under the endogenous islet amyloid polypeptide excretory effect that glucose stimulates, contrasted first intraperitoneal and given glucose (250mg/0.5mL D-glucose; Apart from tube feed t=-30 minute; N=9) to the effect and the observed damage (n=23) in the vehicle treated rat of the gastritis of alcohol-induced.The excision stomach, and as mentioned above behind the ethanol tube feed 30 minutes to damage grading, get blood examination and survey plasma glucose.Detect the injury response in the dextrose treatment rat (n=9) that gives the dense notes of 3mg vein AC 187 altogether in addition.

Give D-glucose in advance, after t=+30 minute, increase plasma glucose to 123mg/dL (by contrast, contrast is 76mg/dL), shown before that it increased endogenous blood plasma islet amyloid polypeptide concentration to 4.8 ± 0.6 pM in fasting SpragueDawley rat, and gastric injury had been kept the score significantly reduce by 18.5 ± 4.6% (P＜0.0005).But preform injection AC187 keeps the score to the damage in the dextrose treatment rat and does not act on.

One of ordinary skill in the art's of the present invention state of the art has all been represented in all publications mentioned in this manual and patent application.All publication and patent applications all are hereby incorporated by, and its degree clearly and is individually pointed out to be hereby incorporated by as each independent publication and patent application.

Although for the purpose of thorough, utilized elaboration and embodiment to describe in detail foregoing invention, it is evident that, can implement some change and modification within the scope of the appended claims.

Claims (17)

1. amylin family peptides, it comprises the ring district of islet amyloid polypeptide and analogue; The alpha helical region of the alpha helical region of at least a portion thyrocalcitonin or its analogue alpha helical region and thyrocalcitonin or its analogue.

2. the peptide of claim 1, wherein said islet amyloid polypeptide behaviour islet amyloid polypeptide and analogue thereof.

3. the peptide of claim 1, wherein said thyrocalcitonin is a salmon calcitonin see calcimar.

4. the peptide of claim 2, wherein said thyrocalcitonin is a salmon calcitonin see calcimar.

5. the peptide of claim 1, it comprises the aminoacid sequence of formula I: Xaa1 X Xaa3 Xaa4Xaa5 Xaa6 Y Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ ID NO:34), wherein

Xaa1 is A, C, hC, D, E, F, I, L, K, hK, R, hR, S, Hse (homoserine), T, G, Q, N, M, Y, W, P, Hyp (oxyproline), H, V or does not exist;

Xaa3 is A, D, E, N, Q, G, V, R, K, hK, hR, H, I, L, M or does not exist;

Xaa4 is A, I, L, S, Hse, T, V, M or does not exist;

Xaa5 is A, S, T, Hse, Y, V, I, L or M;

Xaa6 is T, A, S, Hse, Y, V, I, L or M;

Xaa8 is A, V, I, L, F or M;

Xaa9 is L, T, S, Hse, V, I or M;

Xaa10 is G, H, Q, K, R, N, hK or hR;

Xaa11 is K, R, Q, N, hK, hR or H;

Xaa12 is L, I, V, F, M, W or Y;

Xaa13 is A, F, Y, N, Q, S, Hse or T;

Xaa14 is A, D, E, G, N, K, Q, R, H, hR or hK;

Xaa15 is A, D, E, F, L, S, Y, I, V or M;

Xaa16 is L, F, M, V, Y or I;

Xaa17 is H, Q, N, S, Hse, T or V;

Xaa18 is K, hK, R, hR, H, u (Cit) or n (Orn);

Xaa19 is F, L, S, Hse, V, I, T or does not exist;

Xaa20 is H, R, K, hR, hK, N, Q or does not exist;

Xaa21 is T, S, Hse, V, I, L, Q, N or does not exist;

Xaa22 is F, L, M, V, Y or I;

Xaa23 is P or Hyp;

Xaa24 is P, Hyp, R, K, hR, hK or H;

Xaa25 is T, S, Hse, V, I, L, F or Y;

Xaa26 is N, Q, D or E;

Xaa27 is T, V, S, F, I or L;

Xaa28 is G or A;

Xaa29 is S, Hse, T, V, I, L or Y;

Xaa30 is E, G, K, N, D, R, hR, hK, H or Q;

Xaa31 is A, T, S, Hse, V, I, L, F or Y; With

Xaa32 is F, P, Y, Hse, S, T or Hyp;

Wherein X and Y can produce key, and are the independent residue of selecting with following side chain: thus the chemical bonding formation intramolecularly connection each other of described side chain, for example disulfide linkage; Amido linkage; Can form the alkyl acid and the alkylamine of cyclic lactames; Alkyl aldehydes or alkylogen and alkylamine, but its condensation and being reduced, thus alkylamine or imine linkage formed; Or thereby described side chain can connect formation alkyl, thiazolinyl, alkynyl, ether or thioether bond.

6. the peptide of claim 5, wherein X and Y form disulfide linkage, amido linkage, imine linkage, amine key, alkyl bond and are connected with the intramolecularly of thiazolinyl key.

7. the peptide of claim 5, it further comprises 1,2,3,4,5,6,7,8,9,10,11 or 12 displacement, insertion, disappearance, prolongation and/or derivation and modifies.

8. the peptide of claim 1, it comprises the aminoacid sequence of formula II: Xaa1 Xaa2 Xaa3Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ IDNO:35), wherein

Xaa1 is A, C, D, F, I, K, S, T or does not exist;

Xaa2 is C, D, S or does not exist;

Xaa3 is A, D, N or does not exist;

Xaa4 is A, L, T or does not exist;

Xaa5 is A or S;

Xaa6 is T, A, S or V;

Xaa7 is C, K or A;

Xaa8 is A, V, L or M;

Xaa9 is L or T;

Xaa10 is G, H or Q;

Xaa11 is K, R, Q or hArg;

Xaa12 is L, W or Y;

Xaa13 is A, F, N, Q, S or T;

Xaa14 is A, D, E, G, N, K, Q or R;

Xaa15 is A, D, E, F, L, S or Y;

Xaa16 is L or F;

Xaa17 is H, Q, S or V;

Xaa18 is K, R, hArg, u (Cit) or n (Orn);

Xaa19 is F, L, S or does not exist;

Xaa20 is H, Q or does not exist;

Xaa21 is T, N or does not exist;

Xaa22 is F, L, M, V or Y;

Xaa23 is P;

Xaa24 is P or R;

Xaa25 is T;

Xaa26 is N;

Xaa27 is T or V;

Xaa28 is G;

Xaa29 is S;

Xaa30 is E, G, K or N;

Xaa31 is A or T; With

Xaa32 is F, P or Y.

9. the peptide of claim 8, it further comprises 1,2,3,4,5,6,7,8,9,10,11 or 12 displacement, insertion, disappearance, prolongation and/or derivation and modifies.

10. the peptide of claim 1, it comprises the aminoacid sequence of formula III: Xaa1 Xaa2 Xaa3Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ IDNO:36), wherein

Xaa1 is A, C, F, I, K, S or does not exist;

Xaa2 is C, D or S;

Xaa3 is A, D or N;

Xaa4 is A, L or T;

Xaa5 is A or S;

Xaa6 is T;

Xaa7 is C or K;

Xaa8 is A or V;

Xaa9 is L or T;

Xaa10 is G, H or Q;

Xaa11 is K, R or hArg;

Xaa12 is L;

Xaa13 is A, F, N, S or T;

Xaa14 is A, D, E, G, N, Q or R;

Xaa15 is A, E, F, L, S or Y;

Xaa16 is L;

Xaa17 is H, S or V;

Xaa18 is K, R, hArg, u (Cit) or n (Orn);

Xaa19 is F, L or S;

Xaa20 is H or Q;

Xaa21 is T or N;

Xaa22 is F, L, M, V or Y;

Xaa23 is P;

Xaa24 is P or R;

Xaa25 is T;

Xaa26 is N;

Xaa27 is T or V;

Xaa28 is G;

Xaa29 is S;

Xaa30 is E, G, K or N;

Xaa31 is A or T; With

Xaa32 is F, P or Y.

11. the peptide of claim 11, it further comprises 1,2,3,4,5,6,7,8,9,10,11 or 12 displacement, insertion, disappearance, prolongation and/or derivation and modifies.

12. an amylin family peptides, it comprises the sequence of SEQ ID NO:40-137.

13. the peptide of claim 12, wherein said peptide and SEQ ID NO:40-137 have at least 95% sequence identity.

14. treat eating disorder, insulin resistant, obesity, unusual postprandial hyperglycemia for one kind; The diabetes of any kind comprise I type, II type and gestational diabetes; The method of metabolic syndrome, dumping syndrome, hypertension, hyperlipemia, cardiovascular disorder, hyperlipidaemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and osteoarthritis, it comprises the described peptide of claim 1.

15. treat eating disorder, insulin resistant, obesity, unusual postprandial hyperglycemia for one kind; The diabetes of any kind comprise I type, II type and gestational diabetes; The method of metabolic syndrome, dumping syndrome, hypertension, hyperlipemia, cardiovascular disorder, hyperlipidaemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and osteoarthritis, it comprises the described peptide of claim 5.

16. treat eating disorder, insulin resistant, obesity, unusual postprandial hyperglycemia for one kind; The diabetes of any kind comprise I type, II type and gestational diabetes; The method of metabolic syndrome, dumping syndrome, hypertension, hyperlipemia, cardiovascular disorder, hyperlipidaemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and osteoarthritis, it comprises the described peptide of claim 8.

17. treat eating disorder, insulin resistant, obesity, unusual postprandial hyperglycemia for one kind; The diabetes of any kind comprise I type, II type and gestational diabetes; The method of metabolic syndrome, dumping syndrome, hypertension, hyperlipemia, cardiovascular disorder, hyperlipidaemia, sleep apnea, cancer, pulmonary hypertension, cholecystitis and osteoarthritis, it comprises the described peptide of claim 12.

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