AU669636B2 - Amylin antagonists and agonists - Google Patents

Description

WO 94/26292 PCT/US94/05282 AMYLIN ANTAGONISTS AND AGONISTS Background of the Invention This invention relates to specific amylin analogs which behave as amylin antagonists and agonists, and to their use in the treatment of diabetes mellitus, and hypercalcemia, and the control of food intake.

Amylin, also known as diabetes associated polypeptide (Cooper et al., Proc. Natl. Acad. Sci. USA, 85:7763-7766 (1988)) or islet/insulinoma amyloid polypeptide (Westermark et al., Proc. Natl. Acad. Sci.

USA, 84:3881-3885 (1987)), is a 37-residue polypeptide amide isolated originally from the amyloid-rich pancreas of insulinoma and noninsulin-dependent diabetic (NIDD) patients. It has subsequently been isolated from the normal pancreas of rat (Asai et al., Biochem. Biophys.

Res. Commun., 164:400-405 (1989)). CDNA cloning (Ferrier et al., J. Mol. Endocrinol., 3:R1-R4 (1989)) and immunocytochemical (Lukinius et al., Diabetologia, 32:240-244 (1989)) studies have demonstrated that amylin is synthesized in the islet cells and stored in the islet secretory granules along with insulin. It is cosecreted with insulin (Kanatsuka et al., FEBS Lett., 259:199-201 (1989)). Low quantities of amylin have also been detected in the stomach, intestine, lung and dorsal root ganglion (Asai et al., Biochem. Biophys. Res. Commun., 169:788-795 (1990)); and Ferrier et al., supra).

Biological investigations that followed the isolation of amylin have shown that amylin inhibits basal and insulin-stimulated glucose uptake as well as glycogen synthesis by soleus muscles (Leighton et al., Nature, 335:632-635 (1988)). This peripheral insulin resistance by amylin has also been demonstrated in vivo by euglycemic glucose clamp studies with dogs (Sowa et al., WO 94/26292 PCT/US94/05282 2 Diabetologia, 33:118-120 (1990)) and rats (Molina et al., Diabetes, 39:260-265 (1990)). Furthermore, these investigations in rats showed that amylin attenuated the inhibition of hepatic glucose output by insulin (Molina et al., supra). Based on these observations and the finding that amylin inhibits basal insulin secretion (Ohsawa et 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 noninsulin-dependent diabetes mellitus (NIDDM), commonly known as Type II diabetes mellitus.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemica, elevated blood sugar levels. This disease affects a significant percentage of the population. There are two major categories of diabetes mellitus, commonly referred to as Type I and Type II. In patients with Type I diabetes mellitus, there is a loss of active p-cells in the islets of Langerhans in the pancreas, resulting in low levels of both insulin and amylin. Cooper, Medical Hypothesis, 26:284-288 (1991). Patients with Type I diabetes mellitus who are treated with insulin frequently have a tendency to develop hypoglycemia as a side effect. In patients with Type II diabetes mellitus, there are elevated levels of amylin. Patients with type II diabetes mellitus display varying resistance to the normal biological effects of insulin. Increased levels of amylin, known as hyperamylinemia, have been implicated in causing insulin resistance in a number of model systems, including genetically obese LA/N-cp rats (Huang et al., Hypertension, 19:i-101 i-109 (1992)), genetically obese diabetic yellow mice (Gill et al., Life Sci, 48:703-718 (1991)), dexamethasone induced diabetic rats (Jamal et al., J. Endocrin., 126:425-429 (1990)), streptozocin induced diabetic rats (Inoue et al., t WO 94/26292 PCT/US94/05282 3 Diabetes, 41:723-727 (1992)), and ventromedial hypothalamic lesioned rats and Zucker rats (Tokuyama et al., Endocrinology, 128:2739-2744 (1991)).

Other studies have shown that amylin, like calci can exhibit serum calcium-lowering effects in rats in vivo as well as in cell culture systems (Datta et al., Biochem. Biophys. Res. Commun., 162:876-881 (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 analogs which behave as amylin antagonists and agonists.

In one aspect, the invention features amylin analogs which are linear analogs of biologically active amylin having the following amino acid formula:

R

1 R2-X-A A 9

-A

1 0

-A

1 1 -A -A -A -A -A -A-A 1

-A

1 9

-A-

2

-A

2 1

A

22

-A

23

-Y-Z

wherein: X is a chain of 0-5 amino acids, inclusive, the N-terminal one of which is bonded to R 1 and R 2 Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of which is bonded to Z; Each of R 1 and R 2 independently, is H, C 1

C

12 alkyl methyl), Cg-C 18 aryl phenyl, naphthaleneacetyl), C 1

-C

12 acyl formyl, acetyl, and myristoyl), C 7

-C

18 j 30 aralkyl benzyl), or C 7

-C

18 alkaryl p-methylphenyl);

A

8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb;

A

9 is Thr, Ala, Anb, Aib, Ser, N-Me-Ser, or N-Me-Thr; WO 94/26292 PCT[US94/05282 -4-

A

10 is Gin, Ala, Asn, N-Me-Gin, Gly, Nva, Aib, or Anb; All is Arg, homo-Arg, diethyl-homo-Arg, Lyse-NH-R (where R is H, a branched or straight chain C 1 -Cl 0 aikyl group, or an aryl group) Orn, or Lys;

A

12 is Leu, Ile, 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, Gin, Gly, N-Me-Asn, Nva, Aib, or Anb;

A

15 is Phe, or any aromatic amino acid with or without substituents;

A

16 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;

A

1 is Val, Ile, Aib, Anb, or N-Me-Vai;

A

18 is His, Thr, 3-Me-His, 1-Me-His, P pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyi-homo-Arg, Lys-e-NH-R (where R is H, a branched or straight chain C 1 -Cl 0 alkyl group, or an aryl group), Ala, Aib, Anb, or Orn;

A

19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala;

A

20 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala;

A

21 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva; A 22 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva;

A

23 is Pile, any aromatic amino acid with or without substituents, Leu, Ile, Val, Aib, Anb, Ala, or N-Me-Leu; and Z sNHR 3 or OR 3 wherein R 3 is H, Cl-C 12 alkyl, C 7 -Cl 0 phenylaikyl, C 3

-C

2 0 aikenyl, C 3

C

20 aikinyl, phenyl, or naphthyi.

WO 94/26292 PCT/US94/05282 5 In preferred embodiments, the analogs are antagonists.

In a highly preferred embodiment, the amylin antagonist corresponds to the N-a acetyl derivative of amino acids 8 through 23 of human amylin with an amidated carboxy at the C-terminus, referred to herein as N-a-ac-human amylin (8-23)-NH 2 having the following formula: N-a-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser- Ser-Asn-Asn-Phe-NH 2 (SEQ ID NO:1) In another preferred embodiment, the amylin antagonist has the following formula: N-a-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser- Ser-Asn-Asn-Leu-NH 2 (SEQ ID NO:2) In another aspect, the invention features amylin analogs which are linear analogs of biologically active amylin having the following amino acid formula:

R

1

R

2 -X-A -A-A 3

-A

4

-A

5

-A

6 -A -A 8 -A -AA-A -A 2

-A

1 3

A

1 4

-A

1 5

-A

1 6

-A

1 7

-A

1 8

-A

1 9

-A

2 0

-A-A

2 2

-A

2 3

-Y-Z

wherein X is a chain of 0-5 amino acids, inclusive, the N-terminal one of which is bonded to R 1 and R2; Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of which is bonded to Z; Each of R 1 and R 2 independently, is H,

C

1

-C

12 alkyl, C 6

-C

18 aryl, C 1

-C

12 alyl,

C

7

-C

18 aralkyl, or C 7

-C

18 alkaryl;

A

1 is Lys, Arg, homo-Arg, diethyl-homo-Arg, Lys-E-NH-R (where R is H, a branched or straight chain C 1

-C

10 alkyl group, or an aryl group), or Orn; -L II-- 1 -3 Lj WO 94/26292 PCT[US94/05282 -6-

A

2 is Cys, or Anb;

A

3 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva;

A

4 is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;

A

5 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb;

A

6 is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;

A

7 is Cys, or Anb;

A

8 is Ala, Nal, Thi, Phe, Bth, Pop, N-Me-Ala, Aib, or Anb;

A

9 is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;

A

10 is Gin, Ala, Asn, N-Me-Gin, Gly, Nva, Aib, or Anb; All is Arg, homo-Arg, diethyl-homo-Arg, Lyse-NH-R (where R is H, a branched or straight chain C 1 -Cl 0 alkyl group, or an aryl group) or Orm;

A

12 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;

A

13 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me- Ala, Aib, or Anb;

A

1 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva;

A

15 is Phe, or any aromatic amino acid with or without substitlients;

A

16 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu;

A

17 is Val, Ile, Aib, Anb, or N-Me-Val;

A

18 is His, Thr, 3-Me-His, i-Me-His, pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (where R is H, a branched or straight chain Cl-Cl 0 alkyl group, or an aryl group), Orn, Ala, Aib, or Anb; WO 94/26292 PCT/US94/05282 -7-

A

19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;

A

20 is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb;

A

21 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva;

A

22 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva;

A

23 is Phe, any aromatic amino acid with or without substitutions, Leu, Ile, Val, Aib, Anb, Ala, or N-Me-Leu; and Z is NHR 3 or OR 3 wherein R 3 is H, C 1

-C

12 alkyl, C 7

-C

10 phenylalkyl, C 3

-C

2 0 alkenyl, C 3

C

20 alkinyl, phenyl, or naphthyl.

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 to herein as human amylin (1-23)-NH 2 having the following formula: Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn- Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-NH 2 (SEQ ID NO:3) In another highly preferred embodiment, the amylin analog corresponds to amino acids 1 through 23 of rat amylin, with an amidated carboxy at the C-terminus, referred to herein as rat amylin (1-23)-NH 2 having the following formula: Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn- Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH 2 (SEQ ID NO:4) In yet another highly preferred embodiment, the amylin analog corresponds to the derivative of amino acids 1 through 23 of rat amylin with a-amino normal butyric acid substitutions at positions 2 and 7, and an amidated i~ ii _4 WO 94/26292 PCT/US94/05282 8 carboxy at the C-terminus, referred to herein as [Anb 2 7 rat amylin (1-23)-NH 2 having the following formula: Lys-Anb-Asn-Thr-Ala-Thr-Anb-Ala-Thr-Gln-Arg-Leu-Ala-Asn- Phe-Leu-Val-Arg-Ser-Ser-Asn-Asn-Leu-NH 2 (SEQ ID In another aspect, the invention features a method of treating Type II diabetes mellitus 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, N-a-achuman amylin (8-23)-NH 2 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 of an amylin agonist of the invention in conjunction with a therapeutic amount of insulin.

In still another aspect, the invention features a method of treating hypercalcemia by administering a therapeutic amount of an amylin agonist of the invention.

The compounds of the invention exhibit a broad range of biological activities, including those related to glucose metabolism, calcium levels in the blood, and appetite. Amylin antagonists of the invention attenuate the inhibition by amylin of insulin-stimulated glucose uptake. As a result, the amylin antagonists of the invention act to reduce hyperglycemia resulting from elevated levels of amylin associated with Type II diabetes mellitus. Amylin agonists of the invention inhibit irsulin stimulated glucose 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 mellitus. Amylin agonists of the invention inhibit insulin stimulated glucose

(I

J L WO 94/26292 PCT/US94/05282 -9uptake, 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 mellitus. Amylin agonists of the invention also decrease serum calcium levels, and are therefore useful for treating hypercalcemia. In addition, amylin agonists exhibit an appetite suppressant effect, while amylin antagonists increase appetite. Amylin agonists and antagonists are therefore useful in controlling food intake. For example, amylin agonists are useful for treating problems of overweight.

Many of the compounds of the invention are especially advantageous because they are truncated versions of the natural amylin peptide. The shorter peptide not only facilitates easier synthesis and purification of the compounds, but also improves selectivity and reduces manufacturing procedures and expenses.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description The drawings will first be briefly described.

Drawings Fig. 1 shows the comparison of the primary structures of human amylin (hAMYLIN) and rat amylin (rAMYLIN).

Fig. 2 shows the effect of human amylin, and N-aac-human amylin (8-23)-NH 2 separately and together, on glucose uptake in C 2

C

12 muscle cells.

Fig. 3a and Fig. 3b show the in vivo effects of saline, rat amylin, N-a-ac-human amylin (8-23)-NH2, and N-a-ac-human amylin (8-23)-NH 2 plus rat amylin on plasma WO 94/26292 PCT/US94/05282 10 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)-NH 2 separately, on insulin stimulated glucose uptake in C 2

C

12 muscle cells.

Fig. 5a and 5b show the in vivo effects of saline, rat amylin, human amylin (1-23)-NH 2 and human amylin (1- 23)-NH 2 plus rat amylin on plasma glucose levels, and plasma insulin levels, respectively, in Sprague Dawley rats.

Fig. 6 shows the in vitro effects of rat amylin (1-23)-NH 2 and [Anb 2 7 rat amylin (1-23)-NH 2 separately, on insulin stimulated glucose uptake in C 2

C

12 muscle cells.

Fig. 7a and 7b show the in vivo effects of saline, rat amylin, [Anb 2 7 rat amylin (1-23)-NH 2 and [Anb 2 7 rat amylin (1-23)-NH 2 plus rat amylin on plasma glucose levels, and plasma insulin levels, respectively, in Sprague Dawley rats.

Structure The sequences of naturally occuring human amylin ("hAmylin") and rat amylin ("rAmyli:n") are set forth in Fig. 1. Balasubramaniam et al., Peptides, 12:919-924 (1991). There is a high degree of sequence homology between amylin from these two species. It is believed that in naturally occuring hAmylin and rAmylin, the cysteine residues at positions 2 and 7, present in both species, form an internal disulfide bond, resulting in a cyclic structure.

The amylin analogs of the invention are based upon the biologically active subfragments comprising amino acids 8-23 of hAmylin and rAmylin and derivatives thereof; and upon the biologically active subfragments comprising amino acids 1-23 of hAmylin and rAmylin and WO 94/26292 PCT/US94/05282 11 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 residue is optically active, it is the L-form configuration that is intended unless the D-form is expressly designated. All peptide sequences mentioned herein are written according to the usual convention whereby the N-terminal amino acid is on the left and the 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, yielding -NH-CH(R)-COOR, and -NH- CH(R)-CONHR as the C-terminal residues, respectively.

When the carboxy terminal substituent is -NH 2 the peptide is in the amidated carboxy form.

As set forth above and for convenience in describing this invention, the conventional and nonconventional abbreviations for the various amino acids are used. They are familiar to those skilled ir the art; but for clarity are listed below.

Asp D Aspartic Acid Ala A Alanine Arg R Arginine Asn N Asparagine Cys C Cysteine Gly G Glycine Glu E Glutamic Acid Gln Q Glutamine His H Histidine Ile I Isoleucine Leu L Leucine Lys K Lysine 1.

p WO 94/26292 PCT/US94/05282 12 Met M Methionine Phe F Phenylalanine Pro P Proline Ser S Serine Thr T Threonine Trp W Tryptophan Tyr Y Tyrosine Val V Valine Orn Ornithine Nal 2-napthylalanine Nva norvaline Thi 2-thienylalanine Pcp 4-chlorophenylalanine Bth 3-benzothienyalanine Bip 4,4'-biphenylalanine Tic tetrahydroisoquinoline-3-carboxylic acid Aib aminoisobutyric acid Anb a-aminonormalbutyric acid Dip 2,2-diphenylalanine The compounds of the present invention can be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methane sulfonic, toluene sulfonic, trifluoroacetic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids, such as the hydrohalic acids, hydrochloric acid, sulfuric acid, or phosphoric acid and the like.

Analysis The structure-activity relationships of amylin and amylin analogs were studied both in an in vitro model using a mouse muscle cell line, C 2

C

12 and an in vivo model using Sprague Dawley rats.

WO 94/26292 PCTUS94/05282 13 In the in vitro studies, insulin stimulated the glucose uptake by the C 2

C

12 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 basal glucose uptake by this cell line. Cholera toxin did not have any effect on insulin stimulated glucose uptake but blocked the inhibitory effect of rat amylin.

Several partial sequences of human and rat amylin and their analogs were synthesized and their effects 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, 12:919-924 (1991). The synthetic peptides were 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 on a Waters Model 600 solvent delivery system in conjunction with a U6K injector, Model 481 spectrophotometer and Baseline 810 Data collection software in an IBM-XT computer. Protected amino acid derivatives (Peninsula, CA), synthesis reagents (Applied Biosystems, CA) and solvents (Fischer Scientific, OH) were obtained commercially and used without further purification. Paramethylbenzhydroxylamine (MBHA) resin (0.45 mmol, NH 2 group) was placed in the reaction vessel of the synthesizer and the amino acid derivatives were coupled automatically using the standard program provided by the manufacturers modified to incorporate a double I

-I

-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 1hydroxybenzotriazole esters (4.4 equivalent) to avoid deamidations or lactam formation. At the end of the synthesis the N-a-Boc group was removed, and the peptide resin (1.3 g) was treated with hydrogen fluoride containing dimethylsulfide ml), p-cresol g) and p-thiocresol g) for one hour at -2 to 4 0 C. HF was evacuated and the residue 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 500 ml of distilled water 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 for an additional 30 minutes, the Ph of the solution was adjusted to 5 with acetic acid. The solution was then stirred with anion-exchange resin (AG-3, Cl- form, 10 g wet weight) for 30 minutes, filtered through 0.45 microns filter and pumped into a semipreparative reversed phase column and purified as described in Balasubramaniam et al., Peptides, 12:919-924 (1991). The overall yield of rat and human amylin thus obtained varied between 10-20%.

In Vitro Assays C2C12 cells were cultured at 37 0 C in a humidified CO, atmosphere, in low glucose (1 g/l) DMEM medium containing 20% fetal bovine serum, and 0.5% chick embryo extract (growth medium). Cells were seeded in 75-cm 2 flasks at a density of 1x10 6 cells per flask. When the cells became confluent (3-4 days), they were trypsinized (0.25% trypsin) and washed with growth medium. The final

LA

WO 94/26292 PCT/US94/05282 15 cell pellet was suspended in growth medium and seeded at a density of 2.5-104 cells/well into 24 well plates (16 mm diameter) and allowed to grow to 70% confluence (3 days). To induce fusion, the mononucleated myoblasts 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 medium). Medium was changed one day before the experiment.

2-deoxyqlucose uptake in C 2

C

12 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-glucose (25 mM) DMEM medium. At the end of incubation, cells were washed with PBS and different doses of amylin or amylin analogs (10pM- 10AM) were added and incubated with 2-deoxy-[ 3 H]-glucose (1mM) for 10 min.

Non-carrier-mediated uptake was determined by incubating the cells with cytochalasin B (15 Uptake was terminated by rapidly aspirating the solution, and cells were washed with ice-cold PBS. Cell-associated radioactivity was determined by lysing the cells in 1 M 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 mononucleated myoblasts grew logarithmically and reached 70% confluence by day 3. Fused cells were detected by day 5 and contained >90% multinucleated myotubes by day 9 (6 days in fusion media). In 6-day-old cells there was a increase in glucose uptake in response to insulin compared to a 68-115% increase in 9-day-old cells. These results are similar to earlier observations (Klip et al., Ip WO 94/2,6292 PCT/US94/05282 16 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 L 6 muscle cell line (Beguinot et al., Endocrinology, 18:446-455 (1986)). Because of these findings, and the observation that insulin stimulated glucose uptake in 9day-old cells in a dose-dependent manner, we used 9-dayold C 2

C

12 cells to test the effects of amylin or amylin analogs on insulin-stimulated glucose uptake. The maximal insulin-stimulated response was observed at 100 nM an: remained plateaued at further increasing doses.

The insulin-stimulated glucose uptake in C 2

C

12 myotubes appears to occur mainly through facilitated diffusion because cytochalasin B(15 AM) inhibited >90% of insulinstimulated 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 (22-24 0 C) under 12-hour light/dark cycle with ad lib access to Purina rat chow and water.

Sprague Dawley rats weighing about 300g were fasted overnight (18-22 hrs). Rats were then anesthetized with sodium pentobarbital (40 mg/kg) and catheters were implanted in the jugular vein. Saline (0.1 ml), rat amylin (50 pg) in saline (0.lml) or peptide fragments/analogs (100g) in saline (0.1 ml) were injected through the jugular vein and then flushed with another 0.1 ml of saline. In the cases of studying antagonistic effects, injection of peptide fragments/analogs (1004g) in saline (0.1 ml) were followed 2 min. later with rat amylin (50 gg) in saline (0.1 ml) injection. 30 min. after the injection of the peptides, blood (4-5 ml) was drawn through the jugular vein and collected in heparinized tubes containing aprotinin (10 Plasma was obtained by i WO 94/26292 PCT/US94/05282 17 centrifugation. Plasma glucose and insulin levels were determined by the glucose oxidase method (Model 27 glucose analyzer, Yellow Springs Instruments, Yellow Springs, OH) and a radioimmunoassay kit (Peninsula Laboratories, Belmont, CA), respectively.

Results Referring to Fig. 2, one of the antagonists of the invention, N-a-ac-human amylin(8-23)-NH 2 exhibited no significant effect on insulin stimulated glucose uptake in the in vitro assay when tested separately. Still referring to Fig. 2, the presence of N-a-ac-human amylin (8-23)-NH 2 (IgM) with human amylin consistently shifted the inhibitory dose-response curve of human amylin on insulin stimulated glucose uptake to the right higher concentrations of human amylin), increasing the

IC

50 value from 0.20 nM to 350 nM.

In vivo effects of N-a-ac-human amylin (8-23)-NH 2 were investigated in anesthetized (45 mg/kg) Sprague Dawley rats (-300 g) fasted overnight 20 The following samples were injected via a cannulated jugular vein into individual rats: 100 Al of saline (n rat amylin (50Ag), N-a-ac-human amylin (8-23)-NH 2 (100 Mg), and N-a-ac-human amylin (8-23)-NH 2 (100g) followed 2 min later by rat amylin (50Ag). Thirty minutes after injection, 4-5 ml blood was collected in heparinized tubes from each of the rats and the plasma separated by centrifugation. Plasma glucose and insulin levels were subsequently determined, and te results are set forth in Fig. 3a and 3b, respectively.

Referring to Fig. 3a, rat amylin significantly increased the plasma glucose level compared to the saline control, while N-a-ac-human amylin (8-23)-NH 2 significantly decreased the plasma glucose levels relative to the control, probably by antagonizing the WO 94/26292 PCT/US94/05282 18 effects of endogenous amylin. Still referring to Fig.

3a, N-a-ac-human amylin (8-23)-NH 2 significantly attenuated the elevation of plasma glucose by rat amylin in the rat which received both N-a-ac-human amylin (8- 23)-NH 2 and rat amylin plasma glucose levels were brought down near the control value). The p values in Fig. 3a and 3b, and throughout, refer to values obtained using the ANOVA program with n equal to 5 to 8.

These observations confirm that N-a-ac-human amylin (8-23)-NH 2 is a potent antagonist of human amylin in vitro, and of rat amylin in vivo.

Referring to Fig. 4, human amylin (1-23)-NH 2 inhibited insulin stimulated glucose uptake in the in vitro assay in a manner similar to human amylin. Still referring to Fig. 4, human amylin (1-23)-NH 2 exhibited a dose-response inhibitory effect on insulin-stimulated glucose uptake by C 2

C

12 cells with a potency comparable to that of intact human amylin.

Referring to Fig. 5a, human amylin (1-23)-NH 2 attenuated rat amylin induced hyperglycemia.

Referring to Fig. 6, [Anb 2 7 rat amylin(l-23)-NH 2 inhibited the insulin stimulated glucose uptake in the in vitro assay in a manner qualitatively similar to rit amylin(1-23)-NH 2 Referring to Fig. 6 and Fig. 4, rat amylin (1-23)-NH 2 exhibited a dose-response inhibitory effect on insulin-stimulated glucose uptake by C 2

C

12 cells with a potency comparable to that of intact human amylin.

Still referring to Fig. 6 and Fig. 4, [Anb 2 7 rat amylin (1-23)-NH 2 also exhibited a potency comparable to that of human amylin.

Referring to Fig. 7, [Anb 2 7 rat amylin(1-23)-NH 2 had no significant effect on amylin induced hyperglycemia, but the tendency was in the direction of attenuation.

WO 94/26292 PCT/US94/05282 19 The results obtained together with reported data in the literature are set forth in Table 1 below.

PEPTIDES C 2

C

1 -e ffect on insulin sUimulated glucose uptake 1 ANESTHETIZED R~kS 2 PLASMA GLUCOSE',' plasma Ca 2 1. human axnylin 2. rat amylin ('IRA") inhibits (Fig. 2) inhibits 5 elevates 3 elevates 3 lowered 4 lowered, 3. HA(1-23)-NH 2 inhibits (similar to human amylin) (Fig. 4) 1. lowers basal (Fig. 2. attenuates amylin induced hyperglycemia N. D.

4. RA(I-23)-NH 2 inhibits (similar to human amylin) (Fig. 6) inhibits (similar to human amylin) (Fig. 6) N. D. N. D.

N. D. [Anb 2 7 ]jRA(1-23)-NH, 1. lowers basal (Fig. 7a) 2. no effect on amylin induced hyperglycemia 1. lowers basal (Fig. 3a) 2. attenuates amylin induced hyperglycemia 6. N-a-Ac-HA( (8-23)-NH 2 1. no effect N. D.

2. attenuates ainylin effects (Fig. 2) 1. Present study; 2. effects of 100 ;Lg peptide analogs on basal or 50 jig rat amyLin induced hyperglycemia; 3. M~olina et at., Diabetes.

39:260-265 (1990); and Young et at., Am, J. Physiology, E457-461 (1990); 4. Data et at., Piochen. Blophys. Res, Commu., 162:876-881 (1989); Sheriff et at., Biochim. Bloiphys. Acta, 1136: 219-222 (1992).

WO 94/26292 PCT[US94/05282 21 The agonist or antagonist effect of other amylin analogs of the invention may be determined by the assays described above.

USE

Amylin inhibits 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 II diabetes mellitus, respectively. Since humans with Type II diabetis mellitus have elevated levels of amylin and elevated blood glucose levels, administration of an amylin antagonist of the invention in an amount sufficient to decrease blood glucose levels to normal or clinically acceptable levels provides therapeutic results. Humans with Type I diabetis mellitus have decreased levels of both insulin and amylin, and when treated with insulin have a tendency to develop hypoglycemia. Administration of an amylin agonist of the invention in an amount sufficient 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 the invention decrease serum calcuim levels and may be administered to humans to treat hypercalcemia. Amylin agonists of the invention exhibit an appetite suppressant effect, while amylin antagonists increase appetite. Amylin agonists and antagonists of the invention are therefore useful in controlling food intake. For example, amylin agonists of the invention may be administered for the treatment of obesity.

WO 94/26292 PCT/US94/05282 22 The peptides of the invention may be administered to a human in one of the traditional modes orally, parenterally, transdermally, or transmucosally), or in a sustained release formulation using a biodegradable biocompatible polymer.

WO 94/26292 PCT/US94/05282 23 SEQUENCE LISTING GENERAL INFORMATION:

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A. Balasubramaniam AMYLIN ANTAGONISTS AfD AGONISTS 7 Fish Richardson 225 Franklin Street Boston Massachusetts

U.S.A.

02110-2804 3.5" Diskette, 1.44 Mb IBM PS/2 Model 50Z or MS-DOS (Version WordPerfect (Version 5.1) 08/060,265 12 May 1993 Clark, Paul T.

30,162 00537/078W01 (617) 542-5070 (617) 542-8906 200154 h: INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: N a Ac Ala Thr Gln Arg Leu Ala Asn Phe Leu Val His Ser SerAsn Asn Phe NH 2 10 15 L' p WO 94/26292 PCT/US94/05282 24 INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2: SEQUENCE CHARACTERISTICS: LENGTH: 16 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: N a Ac Ala Thr Gin Arg Leu Ala Asn Phe Leu Val Arg Ser Ser Asn Asn Leu NH 2 10 15 INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 3: SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Lys Cys Asn Thr Ala Thr Cys Ala Thr Gin Arg Leu Ala Asn Phe Leu 10 Val His Ser Ser Asn Asn Phe NHI 2 INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4: SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Lys Cys Asn Thr Ala Thr Cys Ala Thr Gin Arg Leu Ala Asn Phe Leu 10 Val Arg Ser Ser Asn Asn Leu NH 2 INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Lys Anb Asn Thr Ala Thr Anb Ala Thr Gin Arg Leu Ala Asn Phe Leu 10 Val Arg Ser Ser Asn Asn Leu NH 2 Li WO 94/26292 PCT[US94/05282 25 INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: SEQUENCE CHARACTERISTICS: tENGTH: 37 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Lys Cys Aen Thr Ala Thr CyB Ala Thr Gln Arg Leu Ala 10 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser 25 Gly Ser Asn Thr Tyr INFORMATION FOR SEVUENCE IDENTIFICATION NUMBER: SEQUENCE CHARACTERISTICS: LENGTH: 37 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Asn Phe Leu Thr Aen Val Lys Cys Val Arg Gly Ser Asn Thr Ser Ser Aen Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Aen Phe Leu 10 Aen Aen Leu Gly Pro Val Leu Pro Pro Thr Aen Val 25 Tyr What is claimed is:

(F

Claims (3)

1. An amylin analog of the amino acid formula: R 2 -X-A 8 -A 9 -A 1 O-A 1 1 -A 1 2 -A 1 3 -A 1 -A 15 -A 1 6 -A 1 7 -A 8 A 1 9 -A--A 1 A 2 2 -A 2 3 -y-Z wherein: X is a chain of 0-5 amino acids, inclusive, the N-terminal one of which is bonded to R 1 and R 2 Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of which is bonded to Z; Each of R 1 and R 2 1 independently, is H, C 1 C 12 alkyl methyl) C 6 -C 1 8 aryl phenyl, naphtha leneacetyl) Cl-C 12 acyl formyl, acetyl, and myristoyl), C7-C 18 aralkyl benzyl) or C 7 -C 1 8 alkaryl p-methylphenyl); A 8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Thr, Aib, or Anb; A 9 is Thr, Ala, Anb, Aib, Ser, N-Me-Ser, or N-Me-Thr; is Gin, Ala, Asn, N-Me-Gin, Gly, Nva, Aib, or Anb; All is Arg, homo-Arg, diethyl-homo-Arg, Lys- E-NH-R (where R is H, a branched or straight chain C 1 -Cj 0 alkyl group, or an aryl group) Orn, or Lys; A 12 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu; A 13 is Ala, Nal, Thi, k-he, Bth, Pcp, N-Me- Thr, Aib, or Anb; A 14 is Asn, Ala, Gin, Gly, N-Me-Asn, Nva, Aib, or Anb; A 15 is Phe, or any aromatic amino acid with or without substituents; A 16 is Leu, Ile, Val, Aib, Anb, or- N-Me-Leu; WO 94/26292 PCT/US94/05282

27- A 17 is Val, lie, Aib, Anb, or N-Me-Val; A 18 is His, Thr, 3-Me-His, 1-Me-His, p- pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyl-homo-Arg, Lys-E-NH-R (where R is H, a branched or straight chain C 1 -Co 1 alkyl group, or an aryl group,, Ala, Aib, Anb, or Orn; A 19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala; A 20 is Ser, Thr, N-Me-Ser, N-Me-Thr, Aib, Anb, or Ala; A 21 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva; A 2 2 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva; A 2 3 is Phe, any aromatic amino acid with or without substituents, Leu, lie, Val, Aib, Anb, Ala, or N-Me-Leu; and Z is NHR 3 or OR 3 wherein R 3 is H, CI-C 12 alkyl, C 7 -C 10 phenylalkyl, C 3 -C 20 alkenyl, C 3 C 20 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-a-acetyl derivative of amino acids 8 through 23 of human amylin with an amidated carboxy at the C-terminus ("N-a-ac-human amylin (8-23)-NH 2 having the formula: N-a-ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser- Ser-Asn-Asn-Phe-NH 2 or a pharmaceutically acceptable salt thereof. 4. An amylin analog of claim 2 having the amino acid formula: I t I I I I I I I I I I I "1 WO 94/26292 PfCT1US94/05282 28 N-ci-Ac-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-Arg-Ser*- Ser-Asn-Asn-Leu-N1 2 or a pharmaceutically acceptable salt thereof. Ani amylin analog of the amino acid formula: R 2 -X-Al-A 2 -A 3 -A 4 -A 5 -A 6 -A 7 -A 8 -A 9 -Al 0 -A 1 1 -Al 2 -Al 3 Al 4 -A 1 5 -Al 6 -Al 7 -Al 8 -Al 9 -A 20 -A 2 1 -A 2 2 -A 2 3 -Y-z wherein X is a chain of 0-5 amino acids, inclusive, the N-terminal one of which is bonded to R 1 andR2 Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of which is bonded to Z; Each of and R 2 independently, is H, C 7 -C. 1 8 aralkyl, or C 7 -C 1 8 alkaryl; A' is Lys, Arg, homo-Arg, diethyl-homo-Arg, Lys-e-NH-R (where R is H, a branched or straight chain C 1 -CI 0 alkyl group, or an aryl group), or Orn; A 2 is Cys, or Anb; A 3 is Asn, Ala, Gln, Gly, N-Me-Asn, Aib, Anb, or Nva; A 4 is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb; A 5 is Ala, Nal, Thi, Phe, Bthf Pcp, N-Me-Ala, Aib, or Anb; A 6 is Thr, Ser, N-Me-Ser, or N-Me-Thr, Ala, Aib, or Anb; A 7 is Cys, or Anb; A 8 is Ala, Nal, Thi, Phe, Bth, Pcp, N-Me-Ala, Aib, or Anb; A 9 is Thr, Ser, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb; WO 94/26292 PCTUS94/05282

29- A 10 is Gin, Ala, Asn, N-Me-Gin, Gly, Nva, Aib, or Anb; Al1 is Arg, homo-Arg, diethyi-homo-Arg, Lys- E-NH-R (where R is H, a branched or straight chain C 1 -Cl 0 alkyl group, or an aryl group) or Orn; A 12 is Leu, Ile, 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, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva; A 15 is Phe, or any aromatic amino acid with or without substituents; A 16 is Leu, Ile, Val, Aib, Anb, or N-Me-Leu; A 1 is Val, Ile, Aib, Anb, or N-Me-Val; A 18 is His, Thr, 3-Me-His, i-Me-His,f- pyrozolylalanine, N-Me-His, Arg, homo-Arg, diethyi-homo-Arg, Lys-E-NH-R (where R is H, a branched or straight chain C -C 1 aky group, or an aryl group), Orn, Ala, Aib, or t Anb; A 19 is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb; A 0 is Ser, Thr, N-Me-Ser, N-Me-Thr, Ala, Aib, or Anb; A21 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva; A 2 is Asn, Ala, Gin, Gly, N-Me-Asn, Aib, Anb, or Nva; A 23 is Phe, any aromatic amino acid with or without substitutions, Leu, Ile, Val, Aib, Anb, Ala, or N-Me-Leu; and Z is NHR 3 or OR 3 wherein R 3 is H, C 1 -C 12 aikyl, C 7 -C 1 0 phenylaikyl, C 3 -C 2 0 aikenyl, C 3 C 20 alkinyl, phenyl, or naphthyl. or a pharmaceutically acceptable salt thereof. S- WO 94/26292 PCT/US94/05282 30 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)-NH 2 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)-NH 2 n) 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 a-amino normal butyric acid substitutions at positions 2 and 7, and an amidated carboxy at the C- terminus ("[Anb 2 7 rat amylin 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. -i 9 WO 94/26292 PCT/US94/05282 31 The method of claim 9 in which said amylin antagonist is N-a-ac-human amylin (8-23)-NH 2 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 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 1 or claim i Iri I J I