JPH07509008A - Amylin antagonists and agonists - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background of the Amylin Antagonist and Agonist Invention The present invention provides specific antibodies that behave as amylin antagonists and agonists. Mirin analogs and the treatment of diabetes and hypercalcemia, and food Concerning their use in controlling intake.

Diabetes-related polypeptides (Cooper et al., Proceedings Off National Academy Off Sciences in Ninisei ( Proc.

Natl, Acad, Sci, USA), Volume 85 +7763-77 66 (1988)) or pancreatic islets/insulin secretion/amyloid/polypeptides (Westermark et al., Proceedings Off) ・National Academy of Sciences in Ninisei (Pr. oc, Natl, Acad, Sci, USA), Volume 84: 388 Amylin, also known as p. 1-3885 (1987), was originally Enriched in surin secretion and amyloid in non-insulin dependent diabetes (NIDD) patients This is a 37-residue polypeptide amide isolated from human pancreas. Subsequently, it is la The normal pancreas of a cat was also isolated (Asai et al., Biochemical Bioche m, B1ophys.

Res, Commun, ), Vol. 164: pp. 400-405 (1989) ). cDNA cloning (Ferrier et al., Journal O. Molecular Endophrynology (J, Mo1.E ndocr inol, Volume 3: R1-R4 (1989)) and immunocytochemistry (Ru Lukinius et al., Diabetologia (Diabetolo) gia), Vol. 32: 240-244 (1989)) Synthesized within islet cells and stored together with insulin in islet secretory granules was revealed.

It is secreted together with insulin (Kanatsuka et al. FEBS Letters (FEBS Lett), Volume 259: 199- 201 pages (1989)).

Small amounts of amylin have also been detected in the stomach, intestines, lungs and posterior plate ganglia (amylin Asai et al., Biochemical and Biophysical Research Communications (Biochem, B1ophys, Res, Commun, ), Vol. 169, p. 88-795 (1990): and Herrier et al., supra).

Biological studies following amylin isolation revealed that amylin inhibits basal and insulin-stimulated glucose uptake, as well as gene synthesis. (Leighton et al., Natur e), Vol. 335: pp. 632-635 (1988)). The end of this amylin Insulin resistance in the treetops has been reported by Soya et al. Diabetologia), Volume 33: 118-120 (1990)) and rats (Molina et al., Diabetes 39:260-265 (1990)). By clamp test (euglycemic glucose clamp) It has also been revealed in vivo. Additionally, rats These studies show that amylin inhibits insulin-induced hepatic glucose production. (Molina, supra). these views The finding that amylin and amylin suppress basal insulin secretion (Osawa hsawa) et al., Biochemical and Biofincal Research Committee Communications (I3 iochcm, B1ophys, Res, C ommun, ), Vol. 160, pp. 961-967 (1988), Myrin helps with glucose metabolism and non-insulin, commonly known as N-type diabetes. Suggestions that it may play a role in the pathophysiology of dependent diabetes mellitus (NIDDλ1) has been done.

Diabetes mellitus is a chronic hyperglycemia, a disease characterized by elevated blood sugar levels. I am suffering from Xie Wc. This disease affects a significant proportion of the population. usually There are two main categories of diabetes called type ■ and type ■. Type I diabetes In patients, loss of active beta cells in islets of Langerhans cells in the pancreas occurs, resulting in low levels of both insulin and amylin (Cooper (C oper), Medical Hypothes is), Vol. 26, pp. 284-288 (1991)). often with insulin Patients with type I diabetes who are treated tend to develop hypoglycemia as a side effect. . Amylin levels are increased in patients with type n diabetes. diabetes mellitus n type patients exhibit variable resistance to the normal biological effects of insulin. High school Elevated amylin levels, known as mylinosis, are associated with obese LA/N-cp rats. (Huang et al., Hypertension) , Vol. 19 + p. 1-101-i-109 (1992)) Genetic Obesity Sugar Urinary Yellow Mouse (Gill (GHI) et al., Life Science (Life S) ci, ), Vol. 48, pp. 703-718 (1991)), dexamethacin induction Diabetic rats (Jamal et al., Journal of Endo-Fly Nology (J, Endocrin, ), Vol. 126, pp. 425-429 ( (1990)), streptocytosine-induced diabetic rats (Ino ue) et al., Diabetes, Volume 41 Near 23-72 7 (1992)) and rats with lesions in the ventral midline hypothalamus. and lacquer rat (Tokuyama et al., Endofry Noro) Endocrinology, Volume 128: 2739-2744 (1 Insulin resistance has been shown to occur in a number of model systems, including (991)). and are associated with.

Other studies have shown that amylin, like calcitonin, was Shows serum calcium lowering effect in vivo in animals It has been shown (Dotta et al., Biochemical & Official Research-1 Miunikenjins (Biochem, B1 ophys, Res, ComIlun, ), Volume 162, 876-881 (1989)). Amylin has also been shown to act as an appetite suppressant. (B alasubramaniam) et al., Peptides (Peptides), Vol. 12: pp. 919-924 (1991)).

Summary of the invention In general, the present invention relates to agents that behave as amylin antagonists and agonists. The gist of this is the Mirin analog.

- In embodiments, the invention provides the following amino acid formula: % formula % [During the ceremony] X is a chain of 0-5 amino acids, the N-terminal amino acid is connected to R, and R1. matches; Y is a chain containing O-4 amino acids, the C-terminal amino acid of which is attached to Z. ing: Each R3 and R2 is independently H, C+ -CI2 alkyl (e.g. methyl ), C6-Cl3 aryl (e.g. phenyl, naphthalene acetyl), Cl- Cl! Acyl (e.g. formyl, acetyl and myristoyl), Ct-C1 K aralkyl (e.g. benzyl) or C7-C+s alkaryl (e.g. p-methylphenyl); A@ is Ala, Nal, Th1SPheSBthSP ep, N-Me-Thr, Aib or Anb; 80 is Thr, AlaS Anb, AibSSerSN-Me-3er or N-Me-Thr: AI O is Gin, AlaSAsnSN-Me-Gln, Gly, Nva, A ib or Anb; A l is Arg, homo-, Arg, diethyl-homo-A rg, LyS-ε-NH-R (where R is H1 branched or straight chain C, -C ,. alkyl group, or aryl group), Orn or Lys; 71, It is Leu, He, VaL Aib, Anb or N-Me-Leu :p, +3 is Ala, Na1. Th1SPhe, BthSPep, N-Me-Thr, Aib or Anb; A+1 is Asn, Ala, Gln, GlySN-Me-Asn, Nva, Aib or Anb; A IS is Phe or any aromatic amino acid with or without a substituent noic acid.

Ala is Leu, Ile, Val, Aib, Anb or N-Me-L eu;, A, +7 is Val, Ice, Aib, Anb or N-M e-Vat; A18 is His%Thr, 3-Me-His, l-Me-Hi s, β-pyrozolylalanine, N-Me-His, Arg, homoArg, di Ethyl-homo-Arg, Lys-ε-NH-R (where R is H1 branched chain or is a straight chain C, -C,. alkyl group or aryl group), Ala.

Aib, Anb or Orn; A19 is Ser, Thr,, N-Me-5er, N-Me-ThrSAib , Anb or Ala; A20 is Ser, Thr, N-Me-3er, N-Me-Thr, Aib, Anb or Ala; Δ2I is Asn, Δla , Gin, Gly, N-~1e-Asn%A]bSAnb or Nva; A22 is Asn, Ala, Gln, Gay, N-Me-Asn, Ai b, Anb or Nva; A23 is Phe, with or without a substituent Any aromatic amino acid, Leu, lie, \'alSAib, ΔnbSAI a or N-Me-Leu: Z is NHR3 or ORs: where R 3 is HSc, -c,, alkyl, C7-C167znylalkyl, C3-C,.

alkenyl, C, -C2゜alkynyl, phenyl or naphthyl] amylin analog, a biologically active linear analog of amylin with This is the summary.

In preferred embodiments, the analog is an antagonist.

In a more preferred embodiment, the amylin antagonist has a C-terminus N-α acetate of amino acids 8 to 23 of human amylin with midized carboxy Corresponding to the chill derivative, N-α-aC-human amylin (8-23 )-NH2, and the following formula 2 N-a-Ac-Ala-Thr-G in-Arg-Leu-A l a -A 5n-Phe-Lau-Va 1-H1s-5ar-Ser-Asn-Asn -Phe-N') I2 (SEQ ID NO: 1).

In another preferred embodiment, the amylin antagonist is: er-Asn-Asn-Lau-N'H, (SEQ ID NO: 2).

In another embodiment, the invention provides the following amino acid formula:

[In the formula, A chain containing Xll0-5 amino acids whose N-terminal amino acids are R1 and and R2.

Y is a chain containing O-4 amino acids, the C-terminal amino acid of which is attached to Z. ing: Each R0 and R2 is independently H, C, -C,2 alkyl, C6-cIm ali allyl, Cl-CI2 allyl, (:+-C+a aralkyl or Ct-Cu alkyl reel.

, to 1 is Lys, Arg, homo-, Arg, noethyl homo-, Arg,, L ys-ε-NH-R (here, RIIH1 branched chain or straight chain Cl-C+o a (alkyl group, or aryl group), or Orn.

A2 is Cys or Anb: , to 3 is Asn, AlaSGin, GlySN-Me-Asn, Aib, Anb or ItNva; A4 is ThrSSer, N-Me-3erSN-M e-ThrSAla, Aib or 1tAnb;, to 5 is AlaSNal, Th i, PheSBthSPep, N-Me-Ala, Aibmata l;! Anb ;A6 is Thr, Ser, N-Me-3erSN-Me-Thr, Ala SAib or Anb:A7 is (ys or Anb: Aa is Ala, NaL Th1SPheS Bth, PepSN-Me-Ala , Aibma? :l! Anb;, 9 is Thr, Ser, N-Me-5er, N -Me-Thr, Ala, Aib or Anb:, to is G1n5Ala S, Asn, N-Me-Gin, Gly, Nva, Aib or Anb :AH is Arg, homo-A rg, diethyl-homo, herg, Lys-ε -NH-R (where R is H1 branched chain or Cl-C1° alkyl group of 1! , maj2: maa1nol group), mat21maOrn: , 812 is Leu, lie, Val, , ib, Anb is N-Me-L eu:, 13 is 8 la, NaL Thi, Phe, BthS Pcp, N-Me-, Hela, Aibmatl-180b.

A11 is Hesn, Ala, Gin, Gly, N-Me-AsnSAi bSAnb or Nva;, A, Is has Phe or a substituent or Not any aromatic amino acid 9 Ala is Leu, Ile, Val, AibSAnb or N-Me-Leu ;Al1 is \'al, lie, AibSAnb or N-Me-Val;A H8 is His, Thr, 3-Me-His, 1-Me-His, β-pyro Zolylalanine, N-he4C-tois, Arg, homo-, Arg, diethyl Le-homo-ArgSLys-ε-NH-R, where R is H1 branched or straight chain C, -C,. alkyl group or aryl group), Orn.

AlaSAib, or Anb; , 1g is Ser, Thr, N-Me-Ser, N-Me-ThrSAl aSAib or Anb; A20 is Ser, ThrSN-Me-Set, N-M e-ThrSAlaSAib or AnbHA2I is Asn, AlaSGin , Gly, N-Me-Asn, AibSAnb or Nva; AH is As n5AlaSGln, Gly, N-Me-Asn, AibSAnb or Nva; A23 is Phe, an aromatic atom with or without a substituent; amino acid, Lcu, l1eSVal, Aib, Anb, Ala, or N- Me-Leu; Z is NHR+ or OR1;:: and R3 is H, C1 -C+zfurkyl, C7-CH07xnylalkyl, C3-C2゜alkenyl, C5-Cm. Alkynyl, phenyl or naphthyl] amylin analog, a biologically active linear analog of amylin with This is the summary.

In a more preferred example, the amylin analog has an amide at the C-terminus. Corresponds to amino acids 1 to 23 of human amylin, which has a carboquino In the specification, it is called human amylin (1-23)-NH2, and the following formula is more preferable: In another embodiment, the amylin analog has an amidation group at the C-terminus. Corresponds to amino acids 1t to 23 of rat amylin with luboquino, herein It is called rat amylin (1-23)-NHZ and has the following formula: % formula % In yet another preferred embodiment, the amylin analog comprises 2 and Has an α-amino n-butyric acid substitution at the 7th position and an amidated carboxyne at the C-terminus Corresponding to the derivatives of amino acids 1 to 23 of rat amylin, herein [ Anb” 7] rat amyrin (1-23)-NH, and the following formula: % formula % In another embodiment, the invention provides a therapeutic amount of an amylin agonist of the invention. Diabetic efficacy in humans when administered in combination with therapeutic doses of inosrin The gist is how to treat.

In another embodiment, the invention provides therapeutic amounts of an amylin antagonist of the invention. The present invention is characterized by a method for treating type 1 diabetes in humans by administering .

In a more preferable method for treating type III diabetes, N-α-aC-human amyloid (8-23)-NH2 is administered.

In another embodiment, the invention provides a therapeutic amount of an amylin agonist of the invention. The gist is a method of treating hypercalcemia by administering.

The compounds of the present invention affect glucose metabolism, blood Cal/Rum levels, and appetite. Exhibits a wide range of biological activities, including related ones. Amylin Anne of the present invention Agonists reduce insulin-stimulated glucose uptake inhibition by amylin. Jiru. As a result, the amylin antagonist of the present invention It acts to reduce hyperglycemia due to the associated increase in amylin levels. present invention Amylin agonists inhibit insulin-stimulated glucose uptake, Thereby, it tends to increase blood sugar levels. As a result, the present invention Amylin agonists can reduce the hyperglycemia that often accompanies insulin treatment for industrial-type diabetes. It is useful to reduce Amylin agonists of the present invention have insulin-stimulating properties. tend to inhibit glucose uptake, thereby increasing blood sugar levels .

As a result, the amylin agonists of the present invention are useful for insulin treatment of industrial type diabetes. It is useful in reducing the hyperglycemia that often accompanies. Furthermore, the amylin amylina of the present invention agonists also reduce serum calcium levels, thus treating hypercalcemia It is also useful for In addition, amylin antagonists increase appetite while Amylin agonists exhibit appetite suppressing effects. Therefore, Amylin Agonis and amylin antagonists are useful in controlling food intake. example For example, amylin agonists are useful in treating the problem of overweight.

Because the compounds of the present invention are truncated versions of the natural amylin peptide, Particularly advantageous. Short peptides only facilitate synthesis and purification of the compound. It also improves selectivity and reduces manufacturing steps and costs.

Other features and advantages of the invention appear in the following description of preferred embodiments thereof and in the claims. It will be clear from the range.

detailed description First, I will briefly describe the drawings.

drawing Fig. l shows human amylin (hAMYLIN) and rat amylin (rAM A comparison of the -order structure with YLIN) is shown.

Figure 2 shows human amylin and N-a-ac-human amylin (8-23 ) - Glucose uptake of NHt, separately and together, in C2Cl2 muscle cells It shows the effect on the symptoms.

F ig, 3a and Fig, 3b are 1, saline, rat amylin, N-a-ac-human amylin(8-23)-NH2, and N-a-ac- Human amylin: /(8-23)-NH, + rat amylin, each subura Plasma in Spraque Dawley rats In vivo (in v) glucose levels and plasma insulin levels ivo).

Fig. 4 shows human amylin and human amylin(1-23)-NH2, Insulin-stimulated glucose uptake in separate C2C,2 muscle cells This shows the in vitro effect on.

Fig. 5a and Fig. 5b are saline, rat amylin, human human amylin(1-23)-NHz and human amylin(1-23)-N'H , + rat amylin, respectively, in plasma in Svrak-Dawley rats. In vivo effects on glucose and plasma insulin levels show.

Fig. 5 shows rat amylin (1-23)-NHZ and [Anb''] Kratt amylin(1-23)-NHz, respectively, in czc+z muscle cells. Figure 2 shows in vitro effects on insulin-stimulated glucose levels.

F ig, 7 a and Fig, b are 1, saline, rat amylin, [ Anb”7] rat amyrin (1-23)-NHt and [Anb”° fuco Rat amylin (1-23) - NH1 + rat amylin, each sublar Plasma glucose levels and plasma insulin levels in K-Dawley rats. The effects of in vitro treatment on drug levels are shown.

structure Naturally occurring human amylin (“h amylin”) and rat amylin (“r amylin”) The sequence of "Amylin") is published in Fjg, l (Parasubramanium (B ala subrawaniam et al., Peptides, Vol. 12: 91 9-924 (1991)). There is a high correlation between amylin from these two species. There is degree of sequence homology. In naturally occurring h-amylin and r-amylin, The /Stein residues in the 2nd and 7th positions present in both species It is thought that a cyclic structure is formed by forming a ruphide bond.

Amylin analogs of the invention include amino acids 8-2 of h-amylin and r-amylin. Biologically active subfragments consisting of 3 and their derivatives; Organisms consisting of amino acids 1-23 of phosphorus and r-amylin and their derivatives based on biologically active subfragments. Amylin Ana published herein In the log formula, the symbols AX etc. found in the peptide sequences herein; and Ser, Leu, etc. represent amino acid residues. When the amino acid residue is optically active , D-type means an L-type steric structure unless otherwise specified. Everything shown herein The peptide sequence of is written according to normal convention, so that the N-terminus is on the left. The C-terminus is on the right. A short line between two amino acid residues indicates a peptide bond. show.

The -OR or -NHRII substituent on the carboxy terminus of the peptide -NH- as the C-terminal residue, replacing 10H on the terminal amino acid residue, respectively. CH(R)-COOR and -NH-CH(R)-CONHR are provided. The cal When the boxy-terminal substituent is -NH2, the peptide is in the amidated carboquino form. Ru.

As briefly described above and in the present invention, the conventional and Use unconventional abbreviations. These are known to those skilled in the art: but for clarity For this reason, they are included in the list below.

Asp=D=aspartic acid A la = A = alanine , herg-R=arginine Asn=N=asparagine Cys=C=7 stain c1y=c=glycine G1u=E=glutamic acid G1n=Q=glutamine His=H=Histidine 11e = I = Isoleucine Leu = L- = Roynon Lys＝ni-rinn Met=M=methionine Phe=F=phenylalanine Pro=P==proline 5er=S=serine Thr=T=threonine Trp=W=tryptophan Tyr=Y=tyrosine Val=V=valine 0rn==ornithine Na1=2-naphthylalanine Nva = Norvaline Th1=2-chenylalanine Pcp=4-chlorophenylalanine 8th=3-benzochenylalanine Bip-4,4°-biphenylalanine Tic-tetrahydroisoquinoline- 3-carboxylic acid Aib = aminoisobutyric acid Anb-α-aminobutyric acid Djp-2,2-diphenylalanine The compounds of the invention may be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts Therapeutically acceptable substances such as acetic acid, lactic acid, maleic acid, citric acid, and Maltic acid, ascorbic acid, succinic acid, benzoic acid, salicylic acid, methanesulfonic acid , salts of organic acids such as toluenesulfonic acid, trifluoroacetic acid or pamoic acid, etc. and tannic acid or salts of polymers such as carboquinomethylcellulose, and inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid. It is an acid salt.

analysis Structure-activity relationships of amylin and amylin analogs have been demonstrated in mouse muscle cell lines, C. In vitro model using 2Cl2, as well as Suvrak-Dorilla-nt Experiments were conducted in both in vitro and in vivo models using

In in vitro experiments, insulin inhibits glucose uptake by the Ctcu cell line. stimulation by rat amylin (100 pM) in a dose-dependent manner. reduced. However, rat amylin is It showed no effect on base uptake. Cholera toxin is an insulin-stimulating Although it had no effect on glucose uptake, rat amylin Blocked the inhibitory effect.

Some partial sequences of human and rat amylin and their analogs synthesized and their effects were detailed in in vitro and in vitro models. I looked into it thoroughly.

peptide synthesis Human and rat amylin were prepared according to the method of Parasubramaniam et al., supra. (Balasubramaniai et al. , Peptides, Vol. 12: pp. 919-924 (1991) )). Synthetic peptides were characterized by sequence and mass spectrometry analysis, and It was found to be more than 97% pure by analytical reverse phase chromatography. Ta.

Peptide synthesis was performed using an Applied Biosystems model 430A synthesizer. (Applied Biosystem Model 43085ynthes izer). For HPLC, injector (U6K1n projector), Model 48 Tospect Mouth Photometer (Model 481) spectrophotometer) and IBM-XT computer Baselin 810 data collection software (Baselin e 810Data collection software) Waters Model 600 Solvent Delivery System (Waters Model 6005olvent delivery 5yste+*) I went there. Protected amino acid derivatives (Peninsula, potash) Fornia (CA)), synthetic reagents (Applied Biosystems (App) Lied B iosystems), California (CA) and solvents (Fischer 5 scientific ic), Ohio (OH)) was obtained commercially and used without further purification. Ba Lamethylbenzhydroxylamine (MBHA) resin (0,45+mol.

N Using a standard program provided with modifications to the coupling method, the amino acid is Coupling automatically. All amino acids are preformed in pairs of 2, 2 equivalents. Coupling was carried out using a nominal acid anhydride. However, ArgSAsn and and Gin are pre-formed 1-hydrogen to avoid diamidation or lactam formation. Coupled as droxybenzotriazole ester (4,4 equivalents) . At the end of the synthesis, the N-α-BocM was removed and the peptide resin (1,3 g> dimethyl sulfide (-0,8mi'), p-cresol (~0.8g) and hydrogen fluoride (~10/ml) containing p-thiocresol (~0.2g) and p-thiocresol (~0.2g). It was treated at -2 to 4°C for 1 hour. HF was removed and the residue was converted into trimethyl ether. into a sintered filter funnel, washed repeatedly with diethyl ether, and acetic acid (2x It was extracted at 15 ma and freeze-dried. (The obtained unpurified peptide (100 mg), Dissolve in 6M guanidino HCI (6m7'), dilute with 500ml of purified water, H was adjusted to 8 with ammonia. Then, 0.1% potassium ferrinanide (W /V) solution was added gradually with constant stirring until the yellow color did not disappear. Sara After stirring for 30 minutes, the pH of the solution was adjusted to 5 with acetic acid. Then the solution with anion-exchange resin (AC-3, C1 form, wet weight 10 g) for 30 minutes. Stir and filter through a 0.45 micron filter and half into a reverse phase column. pumped, as described by B. alasbramaniam et al. (Purified (B alasubramanium) ) et al., Peptides, Vol. 12, pp. 919-924 (1991 Year)). The overall yield of rat and human amylin thus obtained varies between 10-20%. It moved.

In vitro assay C2C, □ cells were incubated with 20% tallow serum and Low glucose (1g#) DME containing 15% chicken embryo extract (growth medium) Cultured in M medium. Cells were placed in 75-cm flasks at 1 x 106 per flask. We focused on cell density. When the cells become confluent (3-4 days), they are treated with trypnochloride. The cells were treated with chloride (0° 25% tryp/ton) and washed with growth medium. Final cell level cells in growth medium and plated in 24-well plates at a density of 2.5-10" cells/well. (16 mm in diameter) and grown to 70% confluence (3 days). induce fusion To achieve this, the mononucleated lateral blastocytes were added to 10% horse hydrangea instead of 20% FBS (fusion medium). The cells were exposed to serum-containing medium. To avoid premature detachment of cells, add fusion medium each time. By day 9 (day 6 in fusion media), most of the cells have grown fused into a nucleated nodal tube. The medium was filled before the experiment.

The uptake of 2-thioxyglucose in the C2C, □ lateral tube was as described by Klip et al. (Klip et al., Biochemical Journal (Bi Ochem, J.), Vol. 242, pp. 131-136 (1987)). easy To do this, wash the cells with PBS (low phosphate? #IK! Saline), serum-free, high-glue It was incubated in DMEM medium with lucose (25mM) for 5 hours. incu At the end of the incubation, cells were washed with PBS and treated with different doses of amylin or amylin. Milin analog (10 pM-10 μM) was added and 2-deoxy-[3H]- Incubated with glucose (1mM) for 10 minutes. non-carrier-mediated -carrier-mediated) uptake causes the cells to become cytochalasin B (15 μM). Apply the solution quickly. Uptake was terminated by washing and cells were washed with water-cold PBS. . The cells were digested with IM NaOH and an aliquot was neutralized and scintillated. - The radioactivity associated with the cells was determined by counting in a counter.

The protein content of the aliquots was determined by the Lowry method.

After seeding the cells, the undifferentiated mononucleated embryonic cells increased logarithmically and reached 70% confluence by 3 days. Reached. Fused cells were detected by day 5 and by day 9 (day 6 for fused cells)>9 It contained 0% multinucleated nodal tubes. In response to insulin in 9-day-old cells There was a 68-115% increase in glucose uptake in 6-day-old cells, whereas 6-day-old cells There was a 30% increase in These results are similar to the initial findings (clip (K Lip) et al., loc. cit.). Hypoinsulin response of cells up to 6 days of age is probably due to low insulin receptor density and undifferentiated This may be due to the presence of lateral blastocytes (Beguinot et al. , Endocrinology, Volume 18: 446-4 p. 55 (1986)). These findings and the effects of insulin on 9-day-old cells Because of the finding that glucose stimulates glucose uptake in a dose-dependent manner, we , Insulin-stimulated glucose uptake of amylin or amylin analogs Nine-day-old C1CI2 cells were used to test the effect on the disease. best ince Phosphorus-stimulated responses were observed at 1100n, and at increasing doses It remained at Rato. CIC, □ Insulin-stimulated group in the lateral canal Lucose uptake is induced by cytochalasin B (15 μM), which inhibits insulin by cells. - Inhibits stimulatory 2-thioxyglucose uptake by >90% and therefore primarily promotes This appears to be occurring through diffusion.

In Vipo Assay Suvrak Dawley rats used in this experiment (Novick Miller (Ziv) ic Miller), Zelienople, Penn. , PA)) were incubated with Brina rat chow (Puri) under a 12-hour light/dark cycle. na rat chov) and an air-conditioned room with free access to water ( 22-24°C).

Suvrak Dawley rats weighing approximately 300 g were fasted overnight (18-22 hours). I let it happen. Rats were then treated with bentoparbital sodium (40 mg/kg). The animal was anesthetized and a cannula was implanted into the jugular vein. Sailine (0,1m1), Sarah Rat amylin (50 μg) or saline (0,1 ml) in in (0,1 ml) Peptide fragment/analog (100 μg) in 1 mA was injected through the jugular vein; Then, it was flushed with another saline of 0.1 mr. When experimenting with antagonistic effects of peptide fragment/analogue (100 μg) in saline (0.1 mr). Injection: rat amylin (50 μg) in saline (0.1 mff) I went there two minutes later. Thirty minutes after the injection of the peptide, blood (4-5 mi) was injected into the neck. Heparinized tube containing aprotinin (10 μl) pumped through the vein collected inside. Plasma was obtained by centrifugation. Plasma glucose levels and insulin The glucose oxidase method (Model 27, glucose oxidase method) Glucoseanalyzer (Model 27), yellow ・Springs Instruments Inc. (Yellow Springs I) Yellow Springs, Ohio Springs, OH)) and radioimmunoassay kit (Pennino Peninsula Laboratories, Belmont, CA) .

result Referring to FIG. 2, one antagonist of the present invention, N-α-aC-human amyloid (8-23)-NHz, when tested separately in the in vitro assay. There was no significant effect on insulin-stimulated glucose uptake. moreover As shown in FigJ, N-α-ac-human amylin(8-23)-NHz( The presence of human amylin with 1 μM) induces insulin stimulation of human amylin. The dose-response curve for normal glucose uptake is consistently on the right (i.e., high concentration of human amylin) to shift the IC5o value from 0.20 μM to 350 μM. Increased to M.

The in vivo action of N-α-ac-human amylin(8-23)-NHZ is - Svrag-Dawley animals were fasted overnight (220 hours) and anesthetized (45 mg/kg). Experiments were performed on rats (~300 g). Two minutes after rat amylin (50 μg), The following samples were injected into individual rats via the jugular vein through the cannula: (1 ) 100 μm saline (n=5), (2) rat amylin (50 μg), (3) N-α-ac-human amylin(8-23)-NHZ (100 μg) and and (4) N-α-ac-human amylin (8-23)-NHZ (10 (bzg) . Tests in which 4-5 ml of blood was heparinized from each rat 30 minutes after injection. The plasma was collected into a tube and separated by centrifugation. Glucose levels in plasma and in The surin level is subsequently measured and the results are shown in Fig, 3a and Fig, Each will be listed in 3.b.

See F ig, 3a, rat amylin compared to saline control: Significantly increased plasma glucose levels. On the other hand, N-α-aC-human a Myrin(8-23)-NH2 probably antagonizes the action of endogenous amylin. significantly lowered the glucose level in the plasma compared to the control. Sara As shown in Fig. 3a, N-a-ac-human amylin (8-2 3) -NH2 is N-a-ac-human amyrin(8-23)-NH, and In rats that ingested rat amylin, the plasma Significantly reduced the rise in glucose (i.e., controlled glucose levels in the plasma) ). Figs. 3a and 3b and herein The p value is the value obtained using the ANOVA program with n = 5 to 8. shows.

These findings indicate that N-α-aC-human amylin(8-23)-NH, ・Human amylin in vitro and rat amylin in vivo have potential It is clear that he is a strong antagonist.

Referring to Fig. 4, human amylin (1-23)-NH2 is human amylin (1-23)-NH2. Insulin-stimulated glucose uptake in an in vitro assay similar to inhibited. Furthermore, as shown in Fig. 4, human amylin(1-23)-NH 2. Insulin production by C2Cl2 cells compared to that of natural human amylin - Demonstrated a potential dose-response inhibitory effect on stimulated glucose uptake.

Fjg, 5a, human amylin(1-23)-NHz is Reduced phosphorus-induced hyperglycemia.

Referring to FIG. was produced in vitro in the same quantitative manner as rat amylin(1-23)-NH2. Insulin-stimulated glucose uptake was inhibited in the assay. Fig, 5 and Fig. 4, rat amylin(1-23)-NHZ is Insulin-stimulated glucose uptake by C2C1 cells compared to that of amylin showed a potential dose-response inhibitory effect on Furthermore, Figs. 5 and F ig, 4, [Anb”’] rat amylin(1-23)-NH2 also It showed potential compared to that of human amylin.

Referring to Fig. 7, [Anb2°7 Collasoto Amiri:/(1-23)-NH 2 has no significant effect on amylin-induced hyperglycemia, but the trend is It was on the decline.

The results obtained together with the data reported in the publication are listed in Table 1 below.

The agonistic or antagonistic effects of other amylin analogs of the invention include: It can be determined by the assay described above.

Purpose Amylin inhibits insulin-stimulated glucose uptake and glycogen synthesis. harms the liver and increases glucose production. Therefore, insulin against amylin A specific ratio of glucose appears to be necessary to maintain normal plasma glucose levels. be.

Amylin agonists and antagonists of the present invention are type 1 and n type, respectively. It has useful applications in treating diabetes. People who have developed n-type diabetes have an increased The inventive amylin and elevated blood glucose levels Myrin antagonists should be used in patients with normal or clinically tolerated blood glucose levels. administration in amounts sufficient to reduce the . People who develop type I diabetes have decreased insulin and amylin levels. and also have a tendency to develop hypoglycemia when treated with insulin. . The amylin agonist of the present invention is administered to patients with normal or insulin-induced blood glucose levels. sufficient to raise clinically acceptable levels against phosphorus-induced hypoglycemia. Administration in adequate amounts provides therapeutic results.

Amylin agonists of the present invention reduce serum calcium levels and It can be administered to humans to treat urinary tractemia. Amylin agonists of the invention shows appetite suppressant effects, while amylin antagonists increase appetite.

Therefore, the amylin agonists and antagonists of the present invention suppress appetite intake. Useful for controlling For example, the amylin agonists of the present invention can be used in the treatment of obesity. Administer for use.

The peptides of the invention can be administered in one traditional manner (e.g., orally, by injection, intramuscularly, intramuscularly, orally). ), or administered to humans in sustained-release formulations using biodegradable and biocompatible polymers. Ru.

Sequence list (1) General information: (i) Applicant: A. Balasubramani am) (ii) Title of the invention: Amylin antagonist and agonist (5) ) Number of arrays: 7 (tv) 311 address: (A) Recipient Fish & Rich ardson) (B)JS: 225 Franklin Street (225Fr anklin 5tree) (C) City, Boston (D) State Massachusetts (E) Country, United States ( U, S, A,) (F) Postal code: 02110-2804 (v) Computer Readable format: (A) Media type, 3.5” diskette, 1.44M b (B) Computer: I BM-PS/2 Model 50Z or 55SX (C) Operating system: MS-DO8 (Virgitan 5.0) (D) Software: Word Perfect (version 5.1) (vi) Latest application data (A) Application number: (B) Filing date.

(C) Classification: (Axis) Previous application data: (A) Application number: 081060.265 (B) Application date + May 12, 1993 Day (i) Agent information (A) Name Clark, Ball T (C1ark, Paul T.) (B ) Registration number 30.162 (C) Reference/File number 005371078WO1(ix) Telecommunique Noyon information (A) Telephone (617) 542-5070 (B) Fax (617) 54 2-8906 (C) Telex 200154 (2) Information regarding sequence determination number 1 (i) Characteristics of array・ (, to) Array length 16 (B) Sequence type Amino acid (C) Number of towns (D) Topolon - linear (xi) Sequence Sequence number: I N a AC included 111 τhr GLn included rg Lau ^La Asn P ha　Lau　Vii　HLm　Sir　Sir　^Sn8|to Pha　n.

S! 0 1s (2) Information regarding sequence determination number 2 (1) Characteristics of array (A) Array length 16 (B) Sequence type, amino acid (C) Number of chains: (D) Topolo/-@chain (X]) Array Sequence number 2 (2) Information regarding sequence determination number, 3: (1) Sequence characteristics (A) Array length: 23 (B) Sequence type: Amino acid (C) Number of chains・ (D) Topology: @chain (xi) Array Sequence number: 3: (2) Information regarding sequence determination number 4: (]) Sequence characteristics (A) Array length = 23 (B) Sequence type Amino acid (C) Number of chains: (D) Topology straight line (xl) Array Sequence number: 4・ (2) Sequence determination number・Information regarding 5・(1) Sequence characteristics.

(A) Array length: 23 (B) Sequence type Amino acid (C) Number of chains (D) Topology @ Zhenzhou (Sword) Arrangement Sequence number 5 u'-1tnu LJO-1-low Lnj Q ■ Mouth ■ Ri: To ■ In ■ H (Ncnz E-1c/) (1) FIG, 2 peptide, 1 sentence [M] 11 = rat 7milin (50ug) Peptide logarithm [M] FIG. 6 a = Illusion to control p〈05 with vM FIG, 7A b = Runt 72 phosphorus No difference in V 1: lG, 73 a = NF, III: NLTo < 0.0 5 Te 11M ant b = vs. F, no significant difference against 11 -・ll) Anb’”; U, Tona 7mm.

Continuation of front page (51) Int, C1,6 identification symbol Internal office reference number A61K 38100 ADP CO7K 14/47 8318-4HI A61K 37102 ACN

Claims (13)

[Claims] 1. Amino acid formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the ceremony: X is a chain of 0-5 amino acids, the N-terminal amino acid is connected to R1 and R2. Matches: Y is a chain of O-4 amino acids whose C-terminal amino acid is attached to Z: Each R1 and R2 is independently H, C1-C12 alkyl (e.g. methyl) , C0-C18 aryl (e.g. phenyl, naphthalene acetyl), C1-C 12 acyl (e.g. formyl, acetyl, and myristoyl), C7-C 18 aralkyl (e.g. benzyl), or C7-C18 alkaryl (e.g. p-methylphenyl); A8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib or Anb; A9 is Thr, Ala, An b, Aib, Ser, N-Me-Ser or N-Me-Thr; A10 is GI n, Ala, Asn, N-Me-GIn, GIy, Nva, Aib or An b; A11 is Arg, homo-Arg, diethyl-homo-Arg, Lys-E-N H-R (where R is H, a branched or straight chain C1-C10 alkyl group, or is an aryl group), Orn or Lys: A12 is Leu, IIe, Val, Aib, Anb or N-Me-Leu;A 13 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib or Anb; A14 is Asn, Ala, Gln, Gly, N-Me-A sn, Nva, Aib or Anb; A15 may have Phe or a substituent; Any aromatic amino acid that does not have: A16 is Leu, IIe, VaI, Aib, Anb or N-Me-Leu;A 17 is Val, IIe, Aib, Anb or N-Me-Va]; A18 is Hi s, Thr, 3-Me-His, 1-Me-His, β-pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys- ε-NH-R (where R is H, branched or straight chain C1-C10 alkyl group , or aryl group), Ala, Aib, Anb or Orm: A19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, An b or Ala; A20 is Ser, Thr, N-Me-Ser, N-Me-Th r, Aib, Anb or Ala: A21 is Asn, Ala, Gln, GIy, N-Me-Asn, Aib, Anb or Nva; A22 is Asn, Ala, G ln, Gly, N-Me-Asn, Aib, Anb or Nva; A23 is Ph e, aromatic amino acid with or without substituents, Leu, ll e, Val, Aib, Anb, Ala or N-Me-Leu; Z is N HR3 or OR3; where R3 is H, C1-C12 alkyl, C7-C10 Phenyl alkyl, C3-C20 alkenyl, C3-C20 alkynyl, phenyl or naphthyl] An amylin analog represented by or a pharmaceutically acceptable salt thereof. 2. The amylin analog of claim 1, which is an antagonist. 3. formula: [There is an array] and amino acid 8 of human amylin with an amidated carboxy at the C-terminus. Amylin analog according to claim 2, corresponding to the N-α-acetyl derivative of Ishi-23 (N-α-ac-human amylin (8-23)-NH2) or its pharmaceutical Salt that can be tolerated. 4. Amino acid formula: [There is an array] The amylin analog or pharmaceutically acceptable salt thereof according to claim 2, which has the following. 5. Amino acid formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, X is a chain of 0-5 amino acids, the N-terminal amino acid is connected to R1 and R2. matches; Y is a region of 0-4 amino acids, the C-terminal amino acid of which is attached to Z; Each R1 and R2 is independently H, C1-C12 alkyl, C6-C18 ali C1-C12 allyl, C7-C18 aralkyl or C7-C18 alkyl Reel; A1 is Lys, Arg, homo-Arg, diethyl-homo-Arg, Ly s-ε-NH-R (where R is H, branched or straight chain C1-C10 alkyl or aryl group), or Orn: A2 is Cys or Anb; A3 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb. or Nva; A4 is Thr, Ser, N-Me-Ser, N-Me-Thr, A la, Aib or Anb; A5 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib or Anb; A5 is Thr, Ser, N- Me-Ser, N-Me-Thr, AIa, Aib or Anb; A7 is Cys or Anb: A8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib or Anb; A9 is Thr, Ser, N-Me-Ser, N-Me-T hr, Ala, Aib or Anb; Al0 is Gln, Ala, Asn, N-M e-Gln, Gly, Nva, Aib or Anb; A11 is Arg, homo-A rg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, branched chain or straight chain C1-C10 alkyl group, or aryl group), or Orn : A12 is Leu, IIe, Val, Aib, Anb or N-Me-Leu;A 13 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib or Anb; A14 is Asn, Ala, Gln, Giy, N-Me-A sn, Aib, Anb or Nva; A15 may have Phe or a substituent; Any aromatic amino acid that does not have: A16 is Leu, IIe, Val, Aib, Anb or N-Me-Leu;A 17 is Val, IIe, Aib, Anb or N-Me-Val: A18 is Hi s, Thr, 3-Me-Hjs, β-pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (here , R is H, a branched or straight chain C1-C10 alkyl group, or an aryl group ), Orn, AIa, Aib, or Anb; A10 is Ser, ・Thr, N-Me-Ser, N-Me-Thr, Ala, A ib or Anb; A20 is Ser, Thr, N-Me-Ser, N-Me-T hr, Ala, Aib or Anb; A21 is Asn, Ala, Gln, Gly , N-Me-ASn, Aib, Anb or Nva; A22 is Asn, Ala, Gln, GIy, N-Me-Asn, Aib, Anb or Nva; A23 is P he, aromatic amino acid with or without substituents, Leu, I le, Val, Aib, Anb, Ala or N-Mc-Leu; Z is NHR3 or OR3; where R3 is H, C1-C12 alkyl, C7-C1 0 phenylalkyl, C3-C20 alkenyl, C3-C20 alkynyl, phenyl nil or naphthyl] An amylin analog represented by or a pharmaceutically acceptable salt thereof. 6. formula: [There is an array] amino acid 1 of human amylin with an amidated carboxyl at the C-terminus. Amylin according to claim 5 corresponding to 23 to 23. Analog (“human amylin ( 1-23)-NH3'') or a pharmaceutically acceptable salt thereof. 7. formula: [There is an array] amino acid of rat amylin with an amidated carboxyl at the C-terminus Amylin analogs according to claim 5 corresponding to 1 to 23 (“rat amylin”) (1-23)-NH3) or a pharmaceutically acceptable salt thereof. 8. formula: [There is an array] with α-aminobutyric acid substitutions at the 2nd and 7th positions and an amidated carboxylic acid at the C-terminus. Claims corresponding to derivatives of amino acids 1 to 23 of rat amylin having kin Amylin analog according to item 5 [Anb217] rat amylin (1-23) -NH2'') and pharmaceutically acceptable derivatives thereof. 9. administering a therapeutic amount of the amylin antagonist of claim 2 to a human with oral diabetes; A method of treating oral diabetes mellitus in a human, characterized in that: 10. The amylin. The antagonist is N-α-ac-human amylin (8-23 )-NH2. 11. The amylin analog of claim 5, which is a therapeutic amount of an antagonist, is administered orally. A method for treating type I diabetes in humans, characterized in that it is administered to a diabetic human. Law. 12. The amylin analog of claim 5 which is an antagonist in a therapeutic amount is administered at high potency. Hypercalcemia in humans characterized by administration to humans with luciumemia. How to treat. 13. administering a therapeutic amount of an amylin analog of claim 1 or claim 5 to a human; A method for controlling food intake in humans, characterized by: