CA2139651A1 - Amylin antagonists and agonists - Google Patents

Abstract

The invention features amylin analogs which behave as amylin antagonists and agonists. The invention also features the use of the amylin antagonist for the treatment of Type II diabetes mellitus, and the use of the amylin agonists for the treatment of both Type I diabetes mellitus and hypercalcemia. The invention also features the use of amylin antagonists and agonists for the control of food intake.

Description

W0~4/26~ 213 9 6 ~1 PCT~S94/05282 AMYLIN ANTAGONISTS AND AGONISTS

Backqround .of the Invention This invention relates to specific amylin analogs 5 which behave as amylin antagonists and agonis~s, and to th~ir u~e in the treatment of diabetes mellitus/ and hypercalcemia, and ~he control of food intak~.
Amylin, also known as diabetes associated polypeptide (Cooper e~ al., Proc. Natl~ Acad. Sci. USA, 10 85:7763~7766 (1988)) or islet/insulinoma amyloid polypeptide tWestermark et al., Proc. _ tl. Acad. Sci.
USA, 84:3881-3~85 (19~7)), is a 37-residue p~lypeptide amide isolated originally from the amyloid~rich pancreas of insulinoma and noninsulin-dependent diabetic (NIDD) 15 patients. It has subsequently been isolated from the normal pancreas of rat ~Asai et al., Biochem. Bioph~s.
Res. Commun., 164:400-405 (198~)). CDN~ clo~ing (Ferrier et al., J. Mol._ ~ndocrinol., 3:Rl-R4 (1989)) and immunocytochemical (hukinius et al., iabetoloqia, 20 32:240-244 (1989)) studies have de~bnstrated that amylin is synthesized in the islet cells and stor~d in the islet ~ecreto ~ granules along with insulin. It i5 cosecreted with insulin (K~natsuka et al., ~BS ~ett., 259~199-201 : ~1989)~0 Low quanti~ies of amylin have also been 2s deitected in the stomach, intestine, lung and dorsal root ganglion (Asai et al., ~ , 169:788-795 (1990));:and Ferxier et al., supra).
Biologl¢al investigation~ tha~ ~ollowed the isolation o~ amylin have shown that amylin inhibits basal 30 and insulin-stimulated glucose:uptake as well as glycogen . .
synthesis by soleus muscles (Leighton et al., ~, 335:632-635 (1988) j . This peripheral insulin resis~anc~
by amy}in has also been d~3monstrated in vivo by euglycemic g}ucose clamp studies with dogs (Sowa e~ al. "
:

W094/26292 PCT~S94/05~82 ~ ~396~ ~

Diabetoloqia, 33:118-120 ~1~90~) and rats (Molina et al., Diabetes, 39:260-265 (1990)). Furthermore, these investigations in rats showe~ that amylin attenuated the inhibition of hepatic glucose output by insulin (Molina 5 et al., supra). Based on thesè observations and the ~inding that amylin inhibits basal insulin secretion (Ohsawa ~t al., Biochem. Biophys. Res. Commun!, 160:961-967 (1989)), it has been suggested that amylin might play a role in glucose metabolism and the pathophysiology of 10 noninsulin-dependent diabetes mellitus (NIDDN), commonly known as Type II diabetes mellitus.
Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemica, i.e., elevated blood sugar levels. This disease affects a significant 15 percentage of the population. There~are two major categories of diabetes mellitus, commonly referred to as Type I and Type II. In patientæ~with Typ~ I diabetes mellitus, th~re is a loss of acti~e ~-cells in the islets of Langerhans in the pancreas, resulting in low levels of ~o both insulin and amylin. Cooper, Medical Hypothesis, 26:284-288 (1991). Patients with T~pe I diabetes mellitus who are treated with i~sulin frequently have a tendency to d~velop hypoglycemia as a side effect. In patiants with ~ e ~I diabetes mellitus, there are 25 elevated levels o~ amylin. Patients with type II
diabetes mellitus display varying resistance to the normal biological effects of insulin. Increased le~els of amylin, known as hyperamylinemia, ha~e been implicated n,causing insulin resistance in a number of model 30; systems, includinq~genetically obese LA/N-cp rats (Huang et al~ y~ertension, l9:i-lQ1 - i-109 (1992)), genetically obese diabetic yellow mlce (Gill et al., ~ife $ci, ~:703-718~(1991)), dexamethasone induced diabetic ~`
; rats (Jamal et a}., J. Endocrin.~ 6:425~-429 (1990)), 35 s~reptozocin induced~diabetic rats (Inoue et alO, ~ , . ~ ~

~; .

W094/26292 PCT~S94/05252 2139fi~1 Diabetes, 41:723-~27 (1992)), and ventromedial hypothalamic lesioned rats and Zucker rats (Tokuyama Pt al., Endocrinoloqy, 128:2739-2744 (1991)).
Other studies have shown that amylin, like 5 calcitonin, can exhibit serum calcium-lowering effects in rats in vivo as well as in cell culture systems (Datta et al., Biochem. Biophys. Res. Commun., 162:~76-881 ~1989)).
Amylin has also been shown to act as an anorectic agent.
Balasubramaniam et al., Peptides, 12:919_924 (1991)~
Summary of the Invention In general, the invention features amylin analog which behave as amylin antagonists and agonists.
In one aspect, the invention features amylin analogs which are linear analogs of biologically active 15 amylin having the following amino acid formula:

Rl R !X A8 A9-Alo-All-Al2~ 3-Al4-Al5-Al6-Al7 A18_A--A----A

wherein: ~
X is a chain o~ 0-5 amino acids, inclusive, the N-terminal one of which is bond~d to R
and R2;
Y is a chain of 0~4 amino acids, inclusive, thç C-tarminal one of which is bonded to Z;
Each of Rl, and R2, independently, is H, C1-C12 al~yl (e.g., methyl), C6-C18 aryl (e.g., phenyl, naphthaleneacetyl), Cl-C12 acyl (e.g., formyl, acetyl, and myristoyl), C7j ~18 ` ~ 30 ;aralkyl (e.g., ~enzyl), or C7-C18 alkaryl (e.g., p-methylphenyl);
A8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or An~;
A9 is Thr, Ala, Anb, Aib, Ser, N-Me Ser, or N-Ne-Thr;

W094/26~92 PCT~S94/05282 ~ ~ ! f A10 is Gln, Ala, Asn, N-~e-Gln, Gly, Nva, Aib, or Anb;
A11 is Arg, homo-Arg, diethyl-homo~Arg, ~ys-~-NH-R (where R is H, a branched or straight chain Cl-C10 alkyl group, or an aryl group), Orn r or Lys;
A12 is Leu, Ile, Val, Aib, Anb, or N-~e-heu;
A13 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb;
A14 is Asn, Ala, Gln, Gly, N-Me-Asn, Nva, Aib, or Anb;
Al5 is Phe,~ or any aromatic amino acid with - or without substituents;
. A16 is Leu, Ile, Val, Aib, Anb, or N-Me~Leu;
. A17 is Val, Ile, Aib, Anb, or N-M~-Val;
~ : Al3 is:His, Thr, 3-Me-His, l-Me-His, ~-::: pyrozolylalanine, N-Me-His, Arg, hom~-Arg, ~ diethyl~-ho~o-Arg, Lys-~-NH-R (where R is H, a ;~ : branchèd or:straight:chain Cl-C10 alkyl ~ group, or~an:aryl group), ~la, Aib, Anb, or ` Orn; : ~
~ . ~
~- ~ A19 is Ser, Thr, N-Me-S~r, N Me-~hr, Aib, ~nb, ~or~Ala;~
A20 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, 25~ . ~Anb,~or Ala;~
A21 is~ Asn, Ala, Gln,~ Gly,: N-Me-Asn,:Aib, ~:~ Anb,: or~:Nva;~
; A22~is~Asn,:Ala, Gln,:Gly, N ~e-Asn, Alb, ~; : ~nb,~ a=r-Nva;
:~ 30 , ~ . A23-,is~he, any aromatic aml:no acid~with ~or~
` without substituents, Leu, Ile, Yal, Aib, ` Anb,~Ala, or~N-Me-Leu; and : Z~is~NHR3~or OR3;:wherein~R3~;~is H, C1-C
alkyl~ 7-Cl~ phenylalkyl,~C3-C20 alken~l, C3 ; : 35 ~ ~ C20~a1kinyl,;~phenyl,~;:or nap hyl.

,: :

W094/26292 213 9 6 5 1 PCT~S9410i282 In preferred embodim~nts, the analogs are antagonists.
In a hiyhly preferred embodiment, the amylin ar,t~gonist correspond~ to the N-~ acetyl derivative of amino acids 8 through 23 of human amylin with an amidated carboxy at 5 the C-terminus, referred to herein as N-~-ac-human amylin (8-23)~NH2, haYing the following ~ormula:

N-a-Ac-Ala-Thr-Gln-Axg-Leu-~la-~sn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH2 (SEQ ID NO:l) In another preferred embodimen~, the amylin antagonist ~ lQ has the following formula:

N-a-Ac-Ala-Thr-Gln-Arg-Leu-Ala-~sn-Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH2. (SEQ ID NO:2) In another aspect, the invention features amylin analogs which are linear analogs of biologically active 15 amylin having the following amino acid formula:

R
R~!x--Al-A2-A3-A4-As-A6 A7-A8 - A9-Alo~ Al2 .A13_ A14~ s_Al6_A17 A18_A19_~,20-A21-A22-A23-Y Z
wherein X is a chain of 0-5 amino acids, inclusive, th~ N-terminal one of which is bonded to R
and R2;
, Y is a ¢hain of 0-4 amino acids, inclusiY~
the C-terminal one of which is bond~d to Z;
Each of Rl, and R2, independently, is H, Cl-C12 alkyl, C6-C18 aryl, Cl-C12 alyl, C7-C18 aralkyl, or C7~C1~ alkaryl;
A1 is I.ys, Arg, homo Arg, diethyl-homo-Arg, Lys-~-NH-R (where R is H, a branched or straight chain Cl-C10 alkyl ~roup, or an aryl group), or Orn;

W094/26292 PCT~S94/05282 A2 is Cys, or ~n~;
A3 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
A4 is Thr, Ser, N-Me-SPr, N-Me-Thr, ~la, Aib, or Anb;
A5 is Ala, Nal, Thi, Phe, ~th, Pcp, N-Me-Ala, Aib, or Anb;
A6 is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, ;
ox 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-~e-Ser, N-Me-Thr, Ala r Aib or ~nb;
AlO is Gln, Ala, Asn, N-Me Gln, Gly, Nva, Aib, or Anb;
All is Arg, homo-Arg, diethyl-homo-Arg, Lys-~-NH-R (where ~ is H, a branched or straight chain C1-ClO alkyl group, or an aryl group~, ~o or Orn;
A12 is Leu, Ile, Val, Aib, Anb, or N-M~--Leu;
A13 i8 Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala,;Aib t or Anb;
A14 is Asn, Ala, Gln,:Gly, N-~e-Asn, Aib, A~bt or Nva; ~
Als is Phe, or any aromatic amino acid with ~ .
or witho~t substîtuents;
. A16 is Leu, Ile, Val, Aib, Anb, or N~Me-Leu; , A17 is Val, Ile, Aib,~Anb, or N-Me-Val;:
A15 is ~is, Thr, 3-Me-His, l-~e-His, ~-pyrozolylalanine, N-Me~ , ~rg, homo-Arg, diethyl-homo-Arg, Lys-~-NH-R (where R is H, a : branched or straiqht chain C1-C10 alkyl ~ group, or an aryl group),:Orn, Ala, Aib, or Anb; :.

' : :
;
.

W094/26292 PCT~S9q/0~282 (--" 21~9~
.

Al9 is Ser, Thr, N-~e-Ser, N-Me-Thr, Ala, Aib, or Anb;
A20 is Ser, Thr, N-~e-Ser, N-Me-Thr, Ala, Aib, or Anb;
s A21 is Asn, Ala, Glnr Gly, N-Ma-Asn, Aib, Anb, or Nva;
A22 is ~sn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
` A23 is Phe, any aromatic amino acid with or without substltutîons, Leu, Ile, Val, Aib, ~nb, Ala, or N-Me-Leu; and Z is ~ 3 or OR3; wherein R3 is H, C1-C12 - alkyl, C7-C10 phenylalkYl, C3~~20 alkenyl~ ~3 C20 alkinyl, phenyl, or naphthyl.
15 In one highly preferred embodiment, the amylin analog corresponds to amino acids 1 through 23 of human amylin with an amidated carboxy at the C-terminus, referred tQ
h~rein as human amyIin (1-23)-NH2, having the ~ollowing formula:

20 Lys-Cys~Asn-Thr-Ala-Thr-Cys-~la-Thr-Gln Arg-Leu Ala-Asn~
Phe-Leu-Val-Hi -Ser-Ser-Asn-Asn-Phe~-NH2. ~SEQ ID NO:3) In another highly pre~erred embodiment, the amylin analog corresponds to amino a::~ds 1 through 23 of rat amylin, with an amidated carboxy at the C-terminus, ref erred to 25 herein as rat amylin (1-23)-NH2, having the following ~ormula:

Ly~;-Cys-Asn-Thr-Aila-Thr-Cys-Ala-~hr-Gln-Arg-Leu-~la-Asn- I
Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn Leu-NH2. (SEQ ID NO:4 In yet another highly preferred embodiment, the amylin 30 analog c:orresponds to the derivative of amino acids through 23 of rat amylin with ~-amino normal butyric: acid substitutions at positions 2 and 7, and an ~midated -.

W094/26292 PCT~S~4/05282 ~g6~ 8 -carboxy at the C-terminus, referxed to herein as [~nb2~7]
rat amylin (~-~3)-N~2, having the following formula:

Lys-Anb-Asn~Thr-Ala-Thr-Anb-Ala-Thr-&ln~Arg-Leu-Ala-Asn~
Phe-Leu-Val-~rg-Ser-Ser-Asn-Asn-Leu-NH2. (S~Q ID NO:5) In another aspect, the invention features a method of treating Type II diabetes melli~us in a human being by administering a therapeutic amount of an amylin antagonist of the invention. In a highly preferred method of treatment of Type II diabetes mellitus, ~ ac-10 h~man amylin (8-23)-NH2 is adminstered. . ~
In another aspect, the invention features a method -:
of treating Type I diabetes mellitus in a human being by ..
administering a therapeutic amount o~ an amylin agonist o~ the invention in conjunction with a therapeutic amount 15 of insulin.
In still another aspect, the invention features a `;
method of trea.ting hypercalcemia by administering a therapeutic amount o~ an amylin agonist o~ the invantion.
The compounds of the invention exhi~it a broad 20 range of biological activities, including thoce related to glucose metabolism, calcium levels in the blood, and ~ppetite. Amylin antagonists o~ the invention attenuate the inhibition by amylin of insulin-stimulated glucose uptake. As a result, the amylin antagonists of the 25 in~ention act to reduce hyperglycemia resulting ~rom elevated levels of amylin associated with Type II ~;~
diabetes mellitus. Amylin agonists of the invention !
inhi~it insulin stimulated glu ose uptake, thereby .
tending to i~crease blood sugar levels. As a result, the 30 amylin agonists of the invention are useful in reducing :~
the hypoglycemia which frequently accompanies insulin .
tr~atment of Type I diabetes mellitus. Amylin agonists ' .
o~ the invention inhibit insulin stimulated glucose !:
~, t W094/26292 PCT~S94/05282 uptake, thereby tending to increase blood sugar levels.
As a result, the amylin agonists of the invention are useful in reducing the hypoglycemia which frequently accompanies insulin treatment of Type I diabetes 5 mellitus. Amylin agonis~s of the invention also decrease serum calcium levels, and are therefore usef~l for treating hypercalcemia. In addition, amylin agonists exhibit an appetite suppressant ef~ect, while amylin antagonists increase appetite~ Amylin agonists and 10 antagonists are therefore useful in controlling food intake. For example, amylin agonists are use~ul for - treating problems of overweight.
Many o~ the compounds of the invention are especially advanta~eous because they are truncatPd 15 versions of the natural amylin peptide. The shorter peptide not only facilitates easier synthesis and purification of tha compounds, but also improves selectivity and reduces manufacturing procedures and expenses.
Othex features and advantages of the invention will be apparent fr~m the following description o~ the preferr~d em~odiments thereof, and ~rom the claims.

Detailed Descri~tion The drawings will ~irst be briefly described~
2s D~awinas Fig. 1 shows the comparison of the primary structures of human amylin ~hANYLIN) and rat amylin ; I ~rAM~LIN). i ~
Fig. 2 shows the effect of human amylin, and N-~-30 ac-human amylin (8-23)-NH2, separately and toge~her, on glucose uptake;in C2C12 muscle cells. ~ ;
Fig~ 3a and Fig. 3b show the in~vivo effects of saline~ rat amylin, N-~-ac-human amylin (~-23~ NH2, and N-~-ac-human amylin (~-23~ NH2 plus rat amyli~ o~ plasma WOg4/26~92 PCT~S94/05282 965~

glucose levels, and plasma insulin levels, respectively, in Sprague Dawley rats.
Fig. 4 shows the in vitro effect of human amylin and human amylin (1-23)-NH2, separately~ on insulin s stimulated glucose uptake in C2C12 muscle cells.
Fig. 5a and 5b show the in vivo effects of saline, rat amylin, human amylin (1-23)-NH2, and human amylin (1-23)~NH2 plus rat amylin on plasma glucose levels, and plasma insul in levels, respectively, in Sprague Dawley lo rats.
Fig. 6 shows the in vitro effects of rat amyl}n 23)-NH2 and ~Anb2~7] rat amylin (1-23)-NH2, separatelyr on insulin stimulated glucose uptake in C2C12 muscle cells~
Fig. 7a and 7b show the in vivo effects of saline, rat amylin, tAnb2~7] rat amylin (1-23)-NH2, and ~Anb2t7 rat amylin (1-23)-NH~ plus rat amylin on plasma glucose levels, and plasma insulin levels, r~spectively, in Sprague Dawley.rats.

Struçture ~he sequences of naturally occuring human amylin ~"hAmylin") and rat amylin ("rAmylinl') are set forth in Fig~ 1. Balasubramaniam et al., E~E~i~@~ 12:919-924 (1991). There is a high degree of s~quence homology 25 betw~en amylin from the~e two species. It is believed that in naturally occuring hAmylin and rAmylin, the cy teine residues at positions 2 and 7, pre~ent in both ! I` , , species, form an internal disulfide bond, resulting in a cyclic structure.
The amylin analogs of the invention are b~sed upon &
the biologically active subfragments comprising amino acids 8-23 o~ hAmylin and r~mylin and deri~atives thereof; and upon the biologically active subfragments comprising amino acids 1-23 o~ h~mylin and rAmylin and , :
a W094t26~92 21 3 9 6 ~ 1 PCT~S94/05282 derivatives thereof. In the amylin analog formulas set forth herein, the symbols Ax and the like; and Ser, Leu and the like, as found in a peptide sequence herein, stand for amino acid residues. When an amino acid 5 residue is optically acti~e, it is the L-form configuration that is intended unless the D-form is expressly de~i~nated. All peptide s~quences mentioned herein are written according to the usual convention where~y the N-terminal amino a~id is on the left and the 10 C-terminal amino acid is on the right. A short line between two amino acid residues indicates a peptide bond.
An OR or an -NHR substituent on the carboxy terminal end of a peptide replaces the -OH on the carboxy terminal amino acid residue, yie~ding -NH-CH(R)-COOR, and -NH-15 C~(R)-CONffR as the C~terminal residues, respectively.
When the ca~boxy terminal substituant is -NH~, the peptide is in the amidated carboxy form.
As set ~orth abo~e and ~or convenience in describing this invention, the conventional and 20 nonconventional abbre~iations for the various amino acids are used. They are familiar to thos~ skilled in the art;
but for clarity are listed below.

Asp = D = Aspartic Acid Ala = A = Alanine 25 ~rg = R = Arginine A~n = N = Asparagine Cys = C = Cysteine Gl~,= G = Gly~ine Glu = E = Glutamic Acid 30 Gln = Q = Glutamine His = ~ = Histidine Ile = I = IsolsuciRe Leu - L = Leucine Lys = K - Lysine -....

, ' W094/26292 PCT~S94/05282 ~ ~ ! li 96~ ~
-Met = M = Methionine Phe - F = Pheny-lalanine Pro = P = Proline S~r = S = Serine 5 Thr = T = Threonine Trp = W = Tryptophan Tyr = Y = Tyrosine Val = V = Valine Orn = Ornithine o Nal = 2-napthylalanine Nva = norvaline Thi = 2-thienylalanine Pcp = 4-chlorophenylalanine Bth = 3-benzothienyalanine 15 Bip = 4~4'~biphenylalanine Tic = tetrahydroisoquinoline-3-carboxylic acid Aib = aminoisobutyric acid Anb = ~-aminonormalbutyria acid Dip = 2,2-diphenylalanine :
The compounds of the prssent invention can be prowided in the ~orm of pharmaceun cally acceptable salts. Examples of preferred salts are those with therapeutically accep able organic acids, e.g~, acetic, lactic, maleic, citric, malic, ascorbic, succinic, 25 benzoic, salicylic, methane sulfonic, toluene sulfonic, tri~luoroacetic, or pamGic acid, as well as polymeric acids such a ~annic acid or carboxymethyl cellulose, and salts with inorganic acids, such as the hydrohalic acids, , ~e~g~, hydrochloric acid, sulfuric acid, or phosphoric 30 acid and the like.
Analysis The structure-activity relationships of amylin and amylin analogs were s~udied both in an-in vitro model usin~ a mouse muscl~ cell line, C2C12, and an in vivo ~5 model using Sprague Dawley rats.

W094/2~g2 213 9 ~ ~ PCT~S94/0~282 In the in vitro studies, insulin stimulated the glucose uptake by the C2C12 cell line in a dose-dependent manner and this was attenuated by rat amylin (100 pM).
However, rat amylin did not exhibit any effect on the 5 basal glucose uptake by this cell lineO Cholera toxin did not have any effect on insulin stimulated glucose uptake but blocked the inhibitory effect of rat amyli~.
Several partial sequences of human and rat amylin and their analogs were synthesized and their effects 10 investigated in the in vitro and in vivo models.
Peptide Synthesis ~ Human and rat amylin were synthesized according to the procedures set forth in Balasubramaniam et al.~ ;
Peptides, i2:919-924 (1991). The~synthetic peptides were 15 characterized by sequence and~mass spectral analyses, and ~were found to be greater than 97% pure by analytical reversed-phase~chromatography. ;~
Peptide synthesis was accomplished on an Applied Biosystem Model 430A~synthesizer. HPLC was carried out 20 on a Waters Model 600 solven~ del~very system in conjunction with a U6K injectorj Model 48~1 - ~ spectrophotometer`and Baseline~810~Data collection software in an IBM-XT~computer. Protected amino acid deri~atives ;~Peninsula, CA),;synthesis~reagents~(~pplied 25 Biosyst~ms, C~ and~solvents tFischer Scienti~ic, OH) were obtained~commercially~`and~used`~without fuxther purif~ication.~Paramethylbenzhydroxylamine (MBHA) resin (0.45 m~ol, NH2~group) was pla~ed in the reaction vessel !"df the synthës~izer and the amino;~aci~d dèrivatives wére 30 cou]~led~automatioal~ly using~the~standard~program provided~
by~ the manufac~urers modified to incorporate a double ~ ",.,,`,5.~".,.i,t",~

W094l26292 . ~ PCT~S94/05282 ~ 9 6~ 14 - !
coupling procedure. All amino acids were coupled using 2.2 equivalents of preformed symmetrical anhydrides.
Arg, Asn, and Gln, however, were coupled as preformed 1- -hydroxybenzotriazole esters (4.4 equivalent) to avoid 5 deamidation~ or lactam formation. At the end of the synthesis the N-~-Boc group was removed, and the peptide resin (1.3 g) was treated with hydrogen fluoride (-10/ml) containing dimethylsulfide (-0.8 ml3, p-cresol (-0.8 g) and p-thiocresol (-0.2 g) for one hour at -2 to 4C. HF
o was evacuated and the residu2 transferred to a fritted filter funnel with diethyl ether, washed repeatedly with ~ diethyl ether, extracted with acetic acid (2X15 ml) and lyophilized. The crude peptide ~100 mg) thus obtained was dissolved in:6 M guanidine HCl (6 ml), diluted with 15 500 ml of distilled wate~ and the Ph adjusted to 8 with ammonia. A solution of 0.1% potassium ferricyanide ~w/v) was then added gradually with constant stirring until a permanent yellow color persisted. After stirring ~or an additional 30 minutes, the Ph of the solution was 20 adjusted to 5 with acetic acid. The solution was ~hen stirxed with anion-exchange resin (AG 3, Cl- form, 10 g wet weight) ~or 30 minutes, ~iltered through 0.45 microns filter and pumped into a semipreparative reversed pha~
column an~ purified as described in Balasubramaniam et 25 al~ E~i~Ç~ 919-924 (1991)~ The overall yield of rat and human amylin thus obtained varied between 10-20%.

In Vi~ro Assaxs C2C12 cells were cultured at 37C in a humidified 5% C2 atmosphere, in low glucose (1 g/l) DME~ medium 30 con~ining 20% fetal bovine serum, and 0.5% chick embryo ~; extract (gro~ h medium). Cells were seeded in 75-cm2 :~ flasks at a density of 1x106 cells~ per flask. When the cells became~confluent (3-4 days), they were trypsinized (O.25% trypsin) and washed with growth medium. The final ::

W094/26~92 ~13 9 ~ 51 PCT~S94/05282 cell pellet was suspended in growth medium and seeded at a density of 2.5-104 cells/well into ~4 well plates (16 mm diameter) and allowed to grow to 70% confluence (3 days). To induce fusion, the mononucleated myoblasts 5 were exposed to medium containing 10% horse serum instead of 20% FBS (fusion medium). Fusion media was changed every day to avoid the premature detachment of cells and the cells were almost completely fused into multinucleated myotubes by the 9th day (6 days in fusion 10 medium). Medium was changed one day~before the experiment.
- 2-deoxyglucose uptake~in C2C12 myotubes was determined as described in Klip et al., Biochem. J., 242:131-136 (1987). In brief, cells were washed with PBS
(phosphate-buffered saline):and incubated for 5h in the ~serum~free, high-glucos~ (25 mM) DNEM~:medium. At the end of incubation, cells were washed:with PBS and different dos~s of amylin o~ amylin analogs~(lOpM- lO~M) were added and incubated.with 2-deoxy-~3H]-glucose (lmM) for 10 min~
20 Non-carrier-mediated ùptake was determined by incubating the cells with cytochalasin B (15 ~M). Uptake was t~erminated by rapidly aspirating the solution, and cells . , ~ .
were washed with~ice-cold PBS. Cell-associated radioacti~ity waa: determined by lysing;the cells in l 25 NaOH~and the aliquots were neutralized and counted in:a ; : scintillation~counter.~ Protein~content;of the aliquots:
~: ~was determined by~the Lowry~:method.~
After seeding, the undifferentiated mononucleat~d 1.
myoblasts grew logarithmically:and reache~ 70% confluence `
: 30 by day 3. Fused cells~were detected by day 5. and contained~>90%~multinucleated~myotubes~by day 9 (6~days ~!
in :fusion medià)~ In 6-day-old;cells ther~ was a ~0% ~i increase in glucose~uptake in response to insulin e~`
:compared~:to a~68-115% ~increase~in 9-day-old cellsO ;:~These ;35~ results~:are similar to earIier~observations tgliP et al., W094l2629~ PCT~S94/05282 96~

supra). The low insulin response by 6-day-old cells, presumably, is due to the presence of undifferentiated myoblasts with low insulin-receptor density as evident in the L6 muscle cell lin~ (Beguinot et al., Endocrinoloov, 18:446-455 (1986)). Because of these ~indings, and the ob~ervation that insulin stimulated glucose uptake in 3-day-old ~ells in a dose-dependent manner, we used 9-day-old C2C12 cells to test the effects of amylin or amylin analogs on insulin-stimuIated glucose uptake. The .
10 maximal insulin-stimulated response was observed at 100 nM and remained plateaued at further increasing doses.
~ The insulin-stimulated glucose uptake in C2C~2 myotubes appears to occur mainly through ~acilitated diffusion because cytochalasin B(15 ~M) inhibited >90% of insulin-15 stimulated 2-deoxyglucose uptake by the cells.
In_Vivo_Assays Sprague Dawley rats (Zivic`Miller, Zelienople, PA) used in this investigation were housed individually in air-conditioned rooms (22-240Cj under 12-hour light/dark 20 cycle with ad lib access to Purina rat chow and water.
Sprague Dawley rat~ weighing about 300g were fasted overnight (18-22 hræ). Rats were then an~sthetized with sodium pentobarbital (40 mg/kg) and catheters were implanted in the jugular vein. Saline (0.1 ml), rat amylin (50 ~g) in saline tO.lml) or peptide ~ragments~analogs (IOO~g) in saline ~0.1 ml) were injectad throuqh the jugular vein and then flushed with another 0.1 ml of saline. In the cases of studying an~agonisti~ effects, injection of peptide 30 fragments/analogs (l~O~g~ ih saline (0.1 ml) were followed 2 min. later with rat amylin (50 ~g~ in saline (0.1 ml) iniection. 30 min. after the injection of the peptides, blo~d (~-5 ml) was drawn through the jugular vein and collPct.d in heparinized tubes containing 35 aprotinin (10 ~ Plasma:was obtained by ~: :
. ~

W094/26292 21 3 9 ~ 5 ~ PCT~S94/05~82 - l7 -centrifugation. Plasma glucose and insulin levels were determined by th~ glucose oxidase method (Model 27 glucose analyzer, Yellow Sprin~s Instruments, Yellow Springs, OH) and a radioimmunoassay kit (Peninsula s Laboratories, Belmont, CA), respectively.

Results Referring to Fig. 2, one of the antagonists of the invention, N-~-ac-human amylin(8-23)-NH~, exhibited no significant e~fect on insulin stimulated glucose uptake lo in the in vitro assay when test~d separately. Still re~erring to Fig. 2, the pr~sence of N-Q-ac-human amylin (8-23)-NH2 (l~M) with human amylin consistently shifted the inhibitory dose-response curve of human amylin on insulin stimulated glucose uptake to the right (l.e., 15 higher concentrations of human amylin~, increasing the IC50 value from 0.20 nM to 350 nM.
In vivo effec~s of N-~-ac-human amylin~(8-23)-NH~
were investigated in anesthetized (45 mg/kg) Sprague Dawley rats (-300 g) fasted overnight (2 20 h). The 20 following samples were injected via a~cannulated jugular vein into individual rats~ 100 ~1 of saline tn = 5), t2) rat amylin (50~g), (3) N-~-a~-human amylin (8-23)-NH2 (100 ~g~, and ~(4) N-~-ac-human amylin ~8-23)-NH2 (lOO~g) follow~d 2 min later by rat amylin (50~g)O Thirty 25 minu~es after:injection, i-5 ml blood was coll2cted in heparinlzed tubes from each of ~he rats and the plasma separated by c~ntri~ugation. Plasma glucose and insulin levels werq subsequently determined,~and:the results' are set forth in Fig. 3a and 3b, respeoti~ely. .
~ Referring~to Fig~.~3a,~:rat amylin~slgnificant1y increased the~ plasma glucose Ievel compared to the saline : control,:while~N~a-ac-human amylin:~(8-23) NH2 significantly ~decreased;~the~plasma glucose le~els relative to: the control, probably by antagonizing the . , ~ . . . .

:: : ::

W094/26~92 PCT~S94/05~82 396 ~ ~ 18 - ~

effects of endo~enous amylin. Still referring to Fig.
3a, N-~-ac-human amylin (8-23)-NH2 significantly attenuated the elevation of plasma glucose by rat amylin in the rat which received both N-~-ac-human amylin (8-s 23)-NH2 and rat amylin (i.e. plasma glucose level~ were brought down near the control value). The p values in Fig. 3a and 3b, and throughout, refer to values obtained using the ANOV~ pro~ram with n equal to 5 to 8.
These observations confirm that N-a ac-human 10 amylin (8-23)-NH2 is a potent antagonist of human amylin in vitro, and of rat amylin in vivo.
- Ref~rring to Fig. ~, human amylin (1-23)-NH2 inhibited insulin stimulated glucose uptake in the ln vitro assay in a manner similar to human amylin. Still ~5 referring to Fig. 4, human amylin (1-23)-NH2 exhibited a dos~-response inhibitory effect on insulin-stimulated gluco~e uptake by C2C12 cells with a potency comparable to that of intact human amylin.
Referrlng to Fig. 5a, hum~n amylin (1-23)-NH2 20 attenuated rat amylin induced hyperglycemia.
Referring to Fig. 6, [Anb2~7J rat amylin(l-23)-NH2 inhibited the insulin stimulated glucose uptake in the in vitro assay in a manner ~ualitatively similar to rat amylin(l-23)-NH2. Referring to:Fig. 6 and Fig. 4, rat 25 amylin (1-23)-NH2 exhibited a dose-response inhibitory effect on insulin-stimulated glucose uptake by C2C12 cells with a potency comparable to that of intact human amylin.
Still referring to Fig. 6 and Fig. 4, [Anb2~7] rat amylin "~ 23)-NH~ also e~hibited a potency comparable to that of 30 human amylin. .
Referring to Fig. 7, ~Anb2~7] rat ~mylin(1-23~-N~2 had no significant eff~ct on amylin induced hyperglycemia, but the tendency was in th~ direction of ~`
attenuation. : ~ ~

'.

:

The results obtained together with reported data in the literature are set forth in Table 1 below.

WO 94/26~92 PCT/IIS94/05t82 .
3965~ -20-o~` U~
æ ~ .

_ _ ~ ~ o ~ I ~" ~
dO ~
. ~ ~ ~ m ~ o u~ I m ~
a o ~ n ~
-~ o I .a 0 IJ al ~ I ~ 0 --I u e C ~ L ai c ~ e ~
C --I U ~ ~ c 3 ~ 0 ~4 O ~ ?~ O l~ ~ e ~~
1 0 0 0 ~ ~ E C ~ c 0_~ _ ~ nl .s: Sl `
a ~ a a) ~ N Z ~
t ~o /' a o .
o~D oU~ C ~

O I --I --I C --I C~1 C: ~ U
u I ~ m ~ ~a ~a ~ d m OI ~ ~ ~ J~
~ I ~ _l ~ ~ ~ O ~ 0,~
U
1 ~C ~C ~C
~3 1 " N

~m ¦ 4~ E ~¦

N " .
I C I

C ~ N 5N ~
k,~

1~I E ~ --: ~ ~ v h ~ ~ 6 Z ~ N
P~I N ~

WOg4/2629~ 213 9 6 S ' PCT~S94/0~282 The agonist or antagonist effect of other amylin analogs of the invention may be determined by the assays described above.
USE
Amylin i~libits insulin stimulated glucose uptake and glycogen synthesis, and increases the hepatic glucose output. Therefore, it appears that a particular ratio of insulin to amylin is required to maintain the normal plasma glucose levels.
The amylin agonists and antagonists of the invention have useful applications in treating Type I and j!~
II diabetes mellitus, respectively. Since humans with Type II diahetis mellitus have elevated levels of amylin and elevated blood glucose levels, administration of an amylin antagonist o~ the inven~ion in an amount sufficient to decrease blood glucose levels to normal or r clinically acceptable levels provides therapeutic results. Humans with Type I diabekis mellitus have decreased leveils of both insulin and amylin, and when treated with insulin have a tendency to develop hypoglycemia. Administratiorl of an amylin agonist of the invention in an amount su~f icient to increase blood glucose levels to normal or clinically acceptable levels in response to insulin induced hypoglycemia, together with a therapeutic amount of insulin, provides therapeutic results.
Amylin agonists of tha invention de~rease serum calcuim levels and may bei administered to humans to treat hypercalcemia. Amylin agonists o~ the invention exhibi~
an appetite suppressant effect, while amylin an~agonists increase appetite. Amylin agonists and antagonists of the invention are therefore useful in ::ontrolling food intake. For example, amylin agonists of the in~ention may be administered ~for the treatment s~f obesity.

;
:

W094/~6~92 ~965~ PCT~Sg4/05~8~ ~ ~

The peptides of the invention may be administered to a human in one of the traditional modes (e.g., orally, parenterally, transdermally, or transmucosally), or in a sustained release formulation using a biodegradable biocompatible polymer.

i , :

W O 94/26292 ~ ~ 3 g ~ ~ I PCTrUS94105282 :

SEQUENCE I.ISTING
(1) GENBRAL INFORhA~IONl (i) APPLI~ANT~ A. Balas~bramaniam (ii) TITLB OF INnn~NTIONs ~MYLIN ANTAGONISTS AND AGONISTS
(iii) NtnC~5R OF s~QuzNce8s 7 (i~) CORRESPONDE~C8 ADD~S8s ~A) ADDRBSSgæs Fl~h & Richardson (C) CI~YI 225 Franklin Street (D) 8~T~t Massachusatt~
(E) COuNTRys U.S.A.
(F) 8IP~ 02110-2804 (v) COUPUT~R RBADABL~ FOR~s ~.
_ (A) ~DIUN TYP~: 3.5" DLs~ette, 1.44 Mb (8) COMPUTERs IBM PSl2 Model 50Z or 55SX
~C) op~RAT~Na SYSTEM: MS-DOS (Version 5.0) (D) SOFTWARE: WordPerfect (Version 5.1) ~vi) CUF~UDNT ~PP~I Q TION DA~s ~A) APP$ICA~ION NUMB~Rs ~8) FI~IN~ DA5~s (C) CLA5SIFICATION~
~vli) ~RIOR APP~IC~TIO~ DaTAs ~A) ~PP~ICA~ION NU~B~R~ 08/060,265 (B) FI~INO D~T~s 12 May 1993 (viii) ATTORNEY/A OE NT INFO~WaTIONs (A) NAUoes Clark, Paul T.
(B) ~aISTRATION N~nC~gRs 30,162 (C) REF~BNC$/DOCKBT NU~B~Rs 0n537/078WOl ,~
~x) 2~COMMUN~C~TION INFORMATIONs (A) TE~æP~ONEs ~617) 542-5070 .
~B)~ EFASs (617) 542-8906 (C) T21E~s 200154 (2) INFORMATION FOR S~QUENC~ ID~NTIFICA~ION NU~B~
I' I
(i) S~QV~NCB C~ARAC~ISTIC8s (A) L~NaT~S 16 ~B) TYP~s amino acid ~RAND~D~S5s ~D~ TOPO~04Ys Linear : (s~ S~Q~ENC$ ~ S~RIP~ONs ~Q ID NO: l:
N a Ac Ala Thr Gln ~rg ~eu Ala A~n Phe Leu Val His Ser Ser Asn A3~ Phe NH
1~ 2 .

WO 94/2629~ PCT/US94/05282 7,~396~ - 24 -(2) INFORM~TION FOR SEQUEN OE IDENTIFI QTION NUMB~Rs 2:
(i) SEQ~ENCB caARAcTERIsTIcs:
~A) LENGT8s 16 ~B) TYPBs amino acid (C) 8TR~NDEDN~SSs ~D) TCPOLO4Y: Linear ~xi) S~QU~NCE D~SCRIP~IO~s SEQ ID NO: 2: .
N a Ac Ala Thr Gln Arg Leu Ala A~n Phe Leu Val Arg Ser Ser Asn Asn Leu NH2 (2) IN~CR~ATION JOR S~QUENCE IDENTIFICA~ION NUMBLR: 3:
(i) 8EQU~NCE OEARAC5eRISTICSs ~a) L~NaTss 23 (B) TYPJI am$no acid :
~C) STRANDEDN~SSs (D) TOPo~oaYs : Linear (xi) 8LQUENCE D~SCRIPTION: SEQ ID NO: 3:

Ly~ Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 5 ~ : 10 15 Val ~L~ Ser Ser Asn Asn Phe NH2 ` ~ :.

~2) INFOR~ATION FOR 8~QUENCF IDENTIFIcaTIoN NU~Rs 4:
(L) SEQVeNCJ c~ARacTFRIsTIc8s (A) LENGT~s 23 (B) TYPJs amino acid~
~ ~C) ST~ANDEDNESSs : (D) TOPO$O~Ys ~ : Linear , ~
(x ) 8EQUENC6 DESCRIPrIONs ~SEQ ID NO: 4:
~y~Cys Acin Thr Ala Thr Cy~Ala Thr Gln Arg Leu Ala A~n Phe Leu VaI Arg S~r Ser A~n Aen Leu NH2 .
:
: ~2) INFORMATION FOR 8LQUENC~ IDEN~IFIdTION NUM~ER: 5:
(i) SEQUENCB C~ARACT~ISTICS:
(A) ~ENGT~s 23 (B) TYPB~ amino acid (C) 8TRANDEDN~SSs ~ (D) TOPO~O~Y2 Linear :~ ~ (xi) 8E~U~NC~ DESCRIPTION: SEQ ID NO: 5:

Lye Anb Asn Thr Ala Thr Anb Ala Thr Gln Arg Leu:Ala Asn Phe Leu : 5 ~ : : 10 15 Val Arg Sex Ser A3n A~:n Leu NH2 ; ~

. .

WO 94l2629~ PCTrUS94/0~282 (2) INFOR~ATlON FOR SEQU~N OE ~DENTIFICATION NU~BER: 6:
(~) 8EQ~ENC~ Ce~RACTERISTICSs (A) LEN~T~s 37 (a~ TYP~ amino acid (C) STRAND~S~s ~D) TOPOLOar~ Lin~ar (~i) SEQU~NCæ D~SCRIPTION: SEQ ID NO: 6:
Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val ~is Ser Ser A3n Asn ~he Gly Ala Ile Leu Ser Ser Thr Asn Val Gly Ser Asn Thr Tyr `

(2) TNFO~ATION FOR SE~U2N OE ID~NTIF~CATION NUWBE~: 7:
(1) SEQUENCE CE~RA~TBR$STICS:
~A) LEN~T~: 37 (B) T~P~s amino a~id (C) 8TRAND~DNESS:
(D) TOPo~oGy Linear (~i) 8BQUENCE ~ESCRIPTIONs SEQ ID NO: 7:

Ly~ Cy~ Asn Thr Ala Thr Cyf3 Ala Thr Gln Arg Leu Ala Asn Phe Leu Val Arg Ser Ser A~n Asn Leu Gly Pro Val Leu Pro Pro Thr A~n Val Gly Ser ~sn Tbr Tyr ~ :

What is claimed is:

~, ,~
,, ,;

.

. .
.

Claims (5)

1. An amylin analog of the amino acid formula:

wherein:
X is a chain of 0-5 amino acids, inclusive, the N-terminal one of which is bonded to R1 and R2;
Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of which is bonded to Z;
Each of R1, and R2, independently, is H, C1-C12 alkyl (e.g., methyl), C6-C18 aryl (e.g., phenyl, naphthaleneacetyl), C1-C12 acyl (e.g., formyl, acetyl, and myristoyl), C7-C18 aralkyl (e.g., benzyl), or C7-C18 alkaryl (e.g., p-methylphenyl);
A8 is Ala, Nal, Thil Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb;
A9 is Thr, Ala, Anb, Aib, Ser, N-Me-Ser, or N-Me-Thr;

A10 is Gln, Ala, Asn, N-Me-Gln, Gly, Nva, Aib, or Anb;
A11 is Arg, homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH-R (where R is H, a branched or straight chain C1-C10 alkyl group, or an aryl group), Orn, or Lys;
A12 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;
A13 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb;
A14 is Asn, Ala, Gln, Gly, N-Me-Asn, Nva, Aib, or Anb;
A15 is Phe, or any aromatic amino acid with or without substituents;
A16 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;

A17 is Val, Ile, Aib, Anb, or N-Me-Val;
A18 is His, Thr, 3-Me-His, 1-Me-His, .beta.-pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH-R (where R is H, a branched or straight chain C1-C10 alkyl group, or an aryl group), Ala, Aib, Anb, or Orn;
A19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala;
A20 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala;
A21 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
A22 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
A23 is Phe, any aromatic amino acid with or without substituents, Leu, Ile, Val, Aib, Anb, Ala, or N-Me-Leu; and Z is NHR3 or OR3; wherein R3 is H, C1-C12 alkyl, C7-C10 phenylalkyl, C3-C20 alkenyl, C3-C20 alkinyl, phenyl, or naphthyl.
or a pharmaceutically acceptable salt thereof.

2. An amylin analog of claim 1 which is an antagonist.

3. An amylin analog of claim 2 corresponding to the N-.alpha.-acetyl derivative of amino acids 8 through 23 of human amylin with an amidated carboxy at the C-terminus ("N-.alpha.-ac-human amylin (8-23)-NH2") having the formula:
N-.alpha.-ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH2, or a pharmaceutically acceptable salt thereof.

4. An amylin analog of claim 2 having the amino acid formula:

N-.alpha.-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH2, or a pharmaceutically acceptable salt thereof.

5. An amylin analog of the amino acid formula:
wherein X is a chain of 0-5 amino acids, inclusive, the N-terminal one of which is bonded to R1 and R2;
Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of which is bonded to Z;
Each of R1, and R2, independently, is H, C1-C12 alkyl, C6-C18 aryl, C1-C12 alyl, C7-C18 aralkyl, or C7-C18 alkaryl;
A1 is Lys, Arg, homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH-R (where R is H, a branched or straight chain C1-C10 alkyl group, or an 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, Ala, Aib, or Anb;
A5 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb;
A6 is Thr, Ser, N-Me-Ser, or N-Me-Thr, Ala, 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-Ma-Thr, Ala, Aib, or Anb;

A10 is Gln, Ala, Asn, N-Me-Gln, Gly, Nva, Aib, or Anb;
A11 is Arg, homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH-R (where R is H, a branched or straight chain C1-C10 alkyl group, or an aryl group), or Orn;
A12 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;
A13 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb;
A14 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
A15 is Phe, or any aromatic amino acid with or without substituents;
A16 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;
A17 is Val, Ile, Aib, Anb, or N-Me-Val;
A18 is His, Thr, 3-Me-His, 1-Me-His, .beta.-pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH-R (where R is H, a branched or straight chain C1-C10 alkyl group, or an aryl group), Orn, Ala, Aib, or Anb;
A19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;
A20 is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;
A21 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
A22 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;
A23 is Phe, any aromatic amino acid with or without substitutions, Leu, Ile, Val, Aib, Anb, Ala, or N-Me-Leu; and Z is NHR3 or OR3; wherein R3 is H, C1-C12 alkyl, C7-C10 phenylalkyl, C3-C20 alkenyl, C3-C20 alkinyl, phenyl, or naphthyl.
or a pharmaceutically acceptable salt thereof.

6. An amylin analog of claim 5 corresponding to amino acids 1 through 23 of human amylin with an amidated carboxy at the C-terminus ("human amylin (1-23)-NH2") having the formula:

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH2, or a pharmaceutically acceptable salt thereof.

7. An amylin analog of claim 5 corresponding to amino acids 1 through 23 of rat amylin, with an amidated carboxy at the C-terminus ("rat amylin (1-23)-NH2") having the formula:

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH2, or a pharmaceutically acceptable salt thereof.

8. An amylin analog of claim 5 corresponding to the derivative of amino acids 1 through 23 of rat amylin with .alpha.-amino normal butyric acid substitutions at positions 2 and 7, and an amidated carboxy at the C-terminus ("[Anb2,7] rat amylin (1-23)-NH2") having the formula:

Lys-Anb-Asn-Thr-Ala-Thr-Anb-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH2, or a pharmaceutically acceptable salt thereof.

9. A method of treating Type II diabetes mellitus in a human being comprising administering to said human being a therapeutic amount of an amylin antagonist of claim 2.

10. The method of claim 9 in which said amylin antagonist is N-.alpha.-ac-human amylin (8-23)-NH2.

11. A method of treating Type I diabetes mellitus in a human being comprising administering to said human being a therapeutic amount of an amylin analog of claim 5 which is an agonist, and a therapeutic amount of insulin.

12. A method of treating hypercalcemia in a human being comprising administering to said human being a therapeutic amount of an amylin analog of claim 5 which is an agonist.

13. A method of controlling food intake in a human being comprising administering to said human being a therapeutic amount of an amylin analog of claim or

claim 5.