AU739557B2

AU739557B2 - Cyclic peptide analogs of somatostatin

Info

Publication number: AU739557B2
Application number: AU11360/00A
Authority: AU
Inventors: David H. Coy; John E. Taylor
Original assignee: Tulane University; Ipsen Bioscience Inc
Current assignee: Ipsen Pharma SAS; Tulane University
Priority date: 1995-09-29
Filing date: 2000-01-14
Publication date: 2001-10-18
Anticipated expiration: 2016-09-04
Also published as: AU1136000A

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): THE ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND AND BIOMEASURE, INC.

Invention Title: CYCLIC PEPTIDE ANALOGS OF SOMATOSTATIN St..

C

The following statement is a full description of this invention, including the best method of performing it known to me/us: 2 CYCLIC PEPTIDE ANALOGS OF SOMATOSTATIN Background of the Invention Native somatostatin is comprised of both a 14amino acid isoform (somatostatin-14) and a 18-amino acid isoform (somatostatin-28). Heiman, et al., Neuroendocrinology, 45:429-436 (1987). Because of the short half-life of the native somatostatin, various somatostatin analogs have been developed, for the treatment of acromegaly. Raynor, et al., Molecular Pharmacol. 43:838 (1993). Five distinct somatostatin receptors have been identified and characterized. Hoyer, et al., Naunyn-Schmiedeberg's Arch. Pharmacol., 350:441 (1994). Somatostatin produces a variety of effects, including modulation of hormone release, growth hormone, glucagon, insulin, amylin, and neurotransmitter release. Some of these effects have been associated with its binding to a specific somatostatin receptor. For example, the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor ("SSTR-2") (Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al., Am. J. Physiol. 268:G102 (1995)) while the inhibition of insulin has been attributed to the somatostatin type-5 receptor ("SSTR-5") (Coy, et al.

197:366-371 (1993)). It is preferred to have an analog which is selective for the specific somatostatin receptor subtype responsible for the desired biological response, thus, reducing interaction with other receptor subtypes which could lead to undesirable side effects.

3 Summary of the Invention In a first aspect of the present invention there is provided a method of inhibiting the release of insulin in a subject in need thereof, which comprises administering to said subject an effective amount of a peptide of the formula:

RI

Ai-A 2

-A

3

-A

4 -D-Trp-Lys-A 7

-A

8 -Ag-R 3 R2 in which 15 AI is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted; Sis an aromatic amino acid;

A

3 is His or an aromatic amino acid; o. A 4 is His or an aromatic amino acid; 20 A 7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid;

A

9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, C1- 12 alkyl, C7- 20 phenylalkyl, C 11 -2 0 naphthylalkyl, C1-1 2 hydroxyalkyl, 25 C7- 20 hydroxyphenylalkyl, C 11 -2 0 hydroxynaphthylalkyl, or COE 1 where El is C1-1 2 alkyl, C7- 20 phenylalkyl, C 11 20 naphthylalkyl, Ci-1 2 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, or C 11 -2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NHYCH 2 Z where Y is a C1-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and Ag; or a pharmaceutically acceptable salt thereof.

In a second aspect of the present invention there is provided a method of treating type II diabetes in a subject in need thereof, which comprises administering to said subject an effective amount of a peptide of the formula: H:\mrar\ip\K-O\P36702-didoc 14/01/00 -4 Al-A 2

-A

3

-A

4 -D-Trp-LyS-A 7

-A

8

-A

9

-R

3

R

2 in which A, is the D- or L-isomer of Cys;

A

2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid;

A

7 is Thr, Ser, or an aliphatic amino acid;

A

8 is an aromatic amino acid; 15 A9is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, C 1 2 alkyl,

C

7 20 phenylalkyl, Cll-.

20 naphthylalkyl, Cl- 1 2 hydroxyalkyl,

C-

20 hydroxyphenylalkyl, Cll..

20 hydroxynaphthylalkyl, or COE, where El is Cl- 1 2 alkyl, C 7 20 phenylalkyl, Cll..

20 20 naphthylalkyl, C 1 12 hydroxyalkyl, C 7 2 0 hydroxyphenylalkyl, or Cll 1 2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NH-YCH 2 'Z where Y is a Cl- 1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A 1 and A 9 or a pharmaceutically acceptable salt thereof.

In a third aspect of the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a peptide of the formula: Al-A 2

-A

3

-A

4 -D-Trp-Lys-A 7

-A

8

-A

9

-R

3 H: \marar\wip\K-\P6702-divdoc 14/01/00 5 in which AI is the D- or L-isomer of Cys;

A

2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid;

A

7 is Thr, Ser, or an aliphatic amino acid;

A

8 is an aromatic amino acid;

A

9 is the D- or L-isomer of Cys; each of RI and R 2 is, independently, H, C 1 -1 2 alkyl, C7- 2 0 phenylalkyl, C 11 -2 0 naphthylalkyl, C 1 -1 2 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, C 11 -2 0 hydroxynaphthylalkyl, or COE 1 where El is C1- 12 alkyl, C7- 20 phenylalkyl, C 11 -2 0 naphthylalkyl, C 1 -1 2 hydroxyalkyl, C7- 2 0 hydroxyphenylalkyl, 15 or C 11 -2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NHYCH 2 Z where Y is a C1- 12 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and Ag; or a pharmaceutically acceptable salt thereof.

In a fourth aspect of the present invention there is provided a method of imaging cells in vivo containing somatostatin receptors comprising administering a peptide of the formula: Al-A 2

-A

3

-A

4 -D-Trp-Lys-A 7 -A8-A 9

-R

3 R2 in which Ai is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted; As is an aromatic amino acid;

A

4 is His or an aromatic amino acid; H:\marar\wip\K-O\P36702-div.doc 14/01/00 6

A

7 is Thr, Ser, or an aliphatic amino acid;

A

8 is an aromatic amino acid;

A

9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, Ci- 12 alkyl,

C

7 -2 0 phenylalkyl, C 11 20 naphthylalkyl, Ci-1 2 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, C 11 -2 0 hydroxynaphthylalkyl, or COE 1 where El is CI-1 2 alkyl, C7- 20 phenylalkyl,

C

11 20 naphthylalkyl, Ci- 1 2 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, or C 11 -2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NHYCH 2 Z where Y is a C1-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and A 9 or a pharmaceutically acceptable salt thereof, provided that at least one aromatic amino acid of the 15 formula Tyr(I) is present in the peptide; to a subject and detecting the presence of said peptide in said subject.

In a fifth aspect of the present invention there is 20 provided a method of imaging cells in vitro containing somatostatin receptors comprising administering a peptide of the formula:

RI

Ai-A 2

-A

3

-A

4 -D-Trp-Lys -A 7 -A-A9-R 3 R2 in which Ai is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid;

A

7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; H:\marar\wip\K-O\P36702-div.doc 14/01/00 7

A

9 is the D- or L-isomer of Cys; each of RI and R 2 is, independently, H, Cl- 12 alkyl, C7- 20 phenylalkyl, C 11 2 0 naphthylalkyl, CI- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, C 11 2 0 hydroxynaphthylalkyl, or COE 1 where El is Ci- 12 alkyl, C7- 20 phenylalkyl,

C

11 20 naphthylalkyl, Ci- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, or C 11 20 hydroxynaphthylalkyl; and

R

3 is NH 2 or NHYCH 2 Z where Y is a Ci- 12 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and A 9 or a pharmaceutically acceptable salt thereof, provided that at least one aromatic amino acid of the formula Tyr(I), wherein said iodine is radioactive iodine, .125I, 12II or 131I, is present in the peptide; 15 to cells and detecting the presence of said peptide in said cells.

In a sixth aspect of the present invention there is provided the use of a peptide of the formula: R1 Ai-A 2

-A

3

-A

4 -D-Trp-Lys- 7

A

8

-A

9

-R

3 25 R2 in which Ai is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid;

A

7 is Thr, Ser, or an aliphatic amino acid;

A

8 is an aromatic amino acid;

A

9 is the D- or L-isomer of Cys; each of RI and R 2 is, independently, H, C1- 12 alkyl, C7-20 phenylalkyl, C11-2 0 naphthylalkyl, Ci- 12 hydroxyalkyl, H:\marar\wip\K-O\P36702-div.doc 14/01/00 -8a- C7- 2 0 hydroxyphenylalkyl, Cll.

2 0 hydroxynaphthylalkyl, or COE, where El is Cl..

12 alkyl, C7,..

2 0 phenylalkyl, Cll..

2 0 naphthylalkyl, C 1 12 hydroxyalkyl, C7,..

20 hydroxyphenylalkyl, or Cll- 2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NH-YCH 2 -Z where Y is a C 1 l.

12 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof, for inhibiting the release of insulin.

In a seventh aspect of the present invention there is provided the use of a peptide of the formula: Al-A 2

-A

3

-A

4 -D-Trp-Lys-A7,-A 8

-A

9

-R

3 2 0 in which A, is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic .amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid; A7, is Thr, Ser, or an aliphatic amino acid;

A

8 is an aromatic amino acid; A9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, Cl- 1 2 alkyl, C7,..

20 phenylalkyl, Cll..

20 naphthylalkyl, Cl..

12 hydroxyalkyl, C7-..

20 hydroxyphenylalkyl, C 11 20 hydroxynaphthylalkyl, or COE, where El is Cl..

12 alkyl, C7,..

20 phenylalkyl, C 1 ll.

20 naphthylalkyl, Cl..

12 hydroxyalkyl, C7,..

20 hydroxyphenylalkyl, or Cll- 2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NH-Y*CH 2 -Z where Y is a Cl- 1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and H:\marar\Wip\K-O\P36702-divdoc 14/01/00 9 a disulfide bond links the side chains of residues Ai and A 9 or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the release of insulin.

In an eighth aspect of the present invention there is provided a peptide of the formula: R1 Al-A 2

-A

3

-A

4 -D-Trp-Lys-A 7

-A

8

-A

9

-R

3

R

2 *in which 15 Ai is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid; 20 A 7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid;

A

9 is the D- or L-isomer of Cys; each of RI and R 2 is, independently, H, CI- 12 alkyl, C7- 20 phenylalkyl, C 11 2 0 naphthylalkyl, Ci- 12 hydroxyalkyl, 25 C7- 20 hydroxyphenylalkyl, C11-2 0 hydroxynaphthylalkyl, or COE 1 where El is CI- 1 2 alkyl, C 7 -2 0 phenylalkyl, C11-20 naphthylalkyl, C 1 12 hydroxyalkyl, C7-2 0 hydroxyphenylalkyl, or C 11 -2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NHYCH 2 Z where Y is a Ci-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and As; or a pharmaceutically acceptable salt thereof when used to inhibit the release of insulin.

H:\marar\wip\K-O\P36702-div.doc 14/01/00 10 In a ninth aspect of the present invention there is provided the use of a peptide of the formula: Al-A 2

-A

3

-A

4 -D-Trp-Lys-A,-A 8

-A

9

-R

3 in which A, is the D- or L-isomer of Cys;

A

2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted;

**A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid; A7 is Thr, Ser, or an aliphatic amino acid;

A

8 is an aromatic amino acid; Ais the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, Cl- 1 2 alkyl, C7- 2 0 phenylalkyl, Cll- 2 0 naphthylalkyl, Cl...

1 2 hydroxyalkyl, C7-..

20 hydroxyphenylalkyl, Cll..

20 hydroxynaphthylalkyl, or COE, where El is Cl- 1 2 alkyl, C7- 2 0 phenylalkyl, Cll- 2 0 naphthylalkyl, Cl..

12 hydroxyalkyl, C7,..

20 hydroxyphenylalkyl, or Cll..

2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NH-YCH 2 -Z where Y is a Cl 1 1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A 1 and A 9 or a pharmaceutically acceptable salt thereof, for the treatment of type II diabetes.

In a tenth aspect of the present invention there is provided the use of a peptide of the formula: Al-A 2

-A

3

-A

4 -D-Trp-Lys-A,-A 8

-A

9

-R

3 /2 H:\-rar\wip\K-\P36702-liv.d0C 14/01/00 11 in which AI is the D- or L-isomer of Cys;

A

2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid;

A

7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid;

A

9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, Ci- 1 2 alkyl, C7- 2 0 phenylalkyl, C 11 2 0 naphthylalkyl, Ci-1 2 hydroxyalkyl, C7- 2 0 hydroxyphenylalkyl, C 11 -2 0 hydroxynaphthylalkyl, or COE 1 where El is C1-1 2 alkyl, C 7 -2 0 phenylalkyl, C 11 20 naphthylalkyl, C 1 -1 2 hydroxyalkyl, C 7 -2 0 hydroxyphenylalkyl, 15 or C 11 -2 0 hydroxynaphthylalkyl; and

R

3 is NH 2 or NH-YCH 2 'Z where Y is a C1-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and Ag; or a pharmaceutically acceptable salt thereof, 20 in the manufacture of a medicament for the treatment of type II diabetes.

In an eleventh aspect of the present invention there is provided a peptide of the formula:

R

A

1

-A

2

-A

3

-A

4 -D-Trp-Lys-A 7

-A

8 -Ag-R 3 R2 in which Ai is the D- or L-isomer of Cys;

A

2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted;

A

3 is an aromatic amino acid;

A

4 is His or an aromatic amino acid; H:\-arar\wip\K-O\P36702-div.doc 14/01/00 12

A

7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; A9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, Cl..

12 alkyl, C7..

2 0 phenylalkyl, C 1 1.

2 o naphthylalkyl, Cl-.

12 hydroxyalkyl, C7..

2 0 hydroxyphenylalkyl, Cll..

2 0 hydroxynaphthylalkyl, or COE, where El is Cl-1 2 alkyl, C7-.

2 0 phenylalkyl, Cll..

2 0 naphthylalkyl, Cl 1 2 hydroxyalkyl, C7...

20 hydroxyphenylalkyl, or Cll-2.

0 hydroxcynaphthylalkyl; and

R

3 is NH 2 or NH-Y-CH 2 *Z where Y is a Cl..

1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof, when used to treat type II diabetes.

In a twelfth aspect of the present invention there is provided a pharmaceutical composition as described above when used to inhibit the release of insulin.

9.**20 In a thirteenth aspect of the present invention there is provided a pharmaceutical composition as described above when used to treat type II diabetes.

In one embodiment, each of A 3 and As, independently, is Phe, p-X-Phe (where X is a halogen, OH, 0dB 3

CH

3 or NO 2 o-X-Phe (where X is a halogen, OH,

OCH

3

CH

3 or NO 2 pyridyl-Ala, Trp, P-Nal, 2,4-dichloro- Phe, Tyr(I), or F 5 -Phe, A 4 is His, Phe, p-X-Phe (where X is a-halogen, OH, OCH 3 Cl! 3 or NO 2 o-X-Phe (where X is a halogen, OH, OCH 3 1 CH 3 or NO 2 pyridyl-Ala, Trp, fi- Nal, 2,4-dichloro-Phe, Tyr(I), or F.-Phe, A 2 is Asn, Gln, Ala, Aib, Val, Leu, Ile, Nie, Nva, Abu, Phe, p-X-Phe (where X is a halogen, OH,' 0dB 3

CH

3 or NO 2 o-X-Phe (where X is a halogen, OH, 0dB 3

CU

3 or NO 2 pyridyl- H:\mar\.ip\K-O\P36702-div-d0C 14/01/00 13 Ala, Trp, P-Nal, 2,4-dichioro-Phe, TyrCIl), FS-Phe, or is deleted, arnd A 7 is Thr, Ser, Ala, Aib, Val, Leu, Ile, Nie, Nva, or Abu.

In a f urther embodiment, A 9 is Cys, each of A 3 and As, independently, is Phe, p-X-Phe (where X is a halogen, OH, or CH 3 Tyr(I), or Trp, A 4 is His, Phe, p-X-Phe (where X is a halogen, OH, or C1i 3 Tyr(I), or Trp, A 2 is Asn, Gin, or is deleted, and A 7 is Thr or Ser.

In a still further embodiment, A 2 is Asn or is deleted, A 3 is Phe, A 4 is Phe, His, p-X-Phe (where X is a halogen, OH, or CH 3 I Tyr or Trp, A 8 is Phe or p-X-Phe (where X is a halogen, OH, or CH 3 and each of R 1 and R 2 1 independently, is H, and R 3 is NH 2 Below are examples of the peptides of this :::.invention as covered by the above formula:

H

2 -c [Cys-Phe-Phe-D-Trp-Lys-Thr-Phe..<ys]

-NH

2 (Analog I),

H

2 -c (D-Cys-Phe-Phe-D-Trp-Lys-Thir-Phe-.Cys)

-NH

2 (Analog II),

H

2 -c (Cys-Phe-Trp-D-Trp-Lys-Thr-Phe-Cys]

-NH

2

H

2 -c[(D-Cys-Phe-Trp-D-Trp-Lys -Thr-Phe-Cys)

-NH

2

H

2 -c (Cys-Phe-His-D-Trp-Lys-Thr-Phe..Cys]-NH 2

H

2 -c[DCsPeHi--r-y-TrPeCs-NH 2

H

2 ~2-c (Cys-Phe-Phe-D-Trp-Lys-Ser.Phe-.Cys

J-NH

2 (Analog IV),

H

2 -c (D-Cys -Phe-Phe-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -c (Cys-Phe-Trp-D-Trp-Lys-Ser-Phe-Cys]

-NH

2 *(Analog

III),

H

2 -c [D-Cys-Phe-Trp-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -c Cys -Phe-His-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -c[D-Cys-Phe-His-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

-H

2 -c[Cys-Phe-Tyr -D-Trp-Lys-Thr-Phe-Cys)

-NH

2 (Analog VIII),

H

2 -c[D-Cys-Phe-Tyr -D-Trp-Lys-Thr-Phe-CysJ

-NH

2

H

2 -c Cys-Phe-Tyr -D-Trp-Lys-Ser-Phe-Cysj]

-NH

2

H

2 -c[D-Cys-Phe-Tyr -D-'rrp-Lys-Ser-Phe-Cys]J-NH 2 14

H

2 -C Cys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys

-NH

2 (Analog V),

H

2 -c [D-Cys-Asn-Phe-Phe-D-Trp-Lys-Thr -Phe-Cys -NH,

H

2 -C Cys-Asn-Phe-Trp-D-Trp-Lys -Thr-Phe-Cys) -NH 2

H

2 -c [D-Cys-An-Ph-Trp-D-Trp-Lys-Thr-Phe-Cys)

-NH

2

H

2 -C[Cys-Asn-Phe-His-D-Trp-Lys -Thr-Phe-Cys] -NH 2

H

2 -c[D-Cys-Asn-Phe-His--D-Trp-Lys-Thr-Phe-Cys]

-NH

2

H

2 -c[Cys-Asn-Phe-Phe-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -C D-Cys-Asn-Phe-Phe-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -C (Cys-Asn-Phe-Trp-D-Trp-Lys-Ser-Phe-Cys]J-NH 2

H

2 -c (D-Cys-Asn-Phe-Trp-D-Tz-p-Lys-Ser-Phe-Cys]J-NH 2

H

2 -C Cys-Asn-Phe-His-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -C[D-Cys-Asn-Phe-His-D-Trp-Lys-Ser-Phe-Cys]

-NH

2

H

2 -C (Cys-Asn-Phe-Tyr -D-Trp-Lys-Thr-Phe-Cys)

NH

2

H

2 -C [D-Cys-Asri-Phe-Tyr -D-Trp-Lys-Thr-Phe-CyG)

NH

2

,I

H

2 -c[DCys-Asn-Phe-Tyr (I)-D-Trp-Lys-Ser-Phe-Cys]

NH

2

H

2 -C [DCysn-Phe-Tyr ()-D-Trp-Lys- Ser-Phe-Cys J

H

2 -C [Cys-Phe-Tyra-D-Trp-Lys-Thr-Phe-Cys]-NH (Analog VII) 2 -c cys-Phe-Cp-a-D-Trp-Lys-Thr-Phe-Cys]

-NH

2 (Analog I),

H

2 -cCysp-Phe-CPa-D-Trp-Lys-Thr-Phe-Cys] -NH 2 (Analog IX),

H

2 -C (Mp-Phe-Phe-D-Trp-Lys -Thr-Phe-DCys-NH 2 (Analog XI),

H

2 -C Cys-Phe-Tyr-D-Trp-Lys-Thr-Phe--Cys]

-NH

2 (Analog XII),

H

2 -C[Cys-Phe-PHe-D-Trp-Lys-Thr-Pyr-CysJ

-NH

2 (Analog XII), H: \Mnrar\.ip\K-O\P36702-div-dOc 14/01/00 15 With the exception of the N-terminal amino acid, all abbreviations Ala or A 2 of amino acids in this disclosure stand for the structure of -NH-CH(R)-CO-, wherein R is a side chain of an amino acid CH 3 for Ala). For the N-terminal amino acid, the abbreviation stands for =N-CH(CH 2 SH)-CO- if it is the D- or L-isomer of Cys or

=C(-CH

2 SH)-CO- if it is the D- or L-isomer of Mpa, wherein R is a side chain of an amino acid. Nle, Nva, pyridyl-Ala, F 5 -Phe, 2,4-dichloro-Phe, P-Nal, Abu, Mpa, Cpa, and Aib are respective abbreviations of the following a-amino acids: norleucine, norvaline, ppyridyl-alanine, pentafluoro-phenylalanine, 2,4- ,dichlorophenylalanine, P-napthylalanine, a-aminobutyric acid, mercaptopropionic acid, p-chlorophenylalanine, and 15 a-aminoisobutyric acid. Tyr(I) refers to an iodinated tyrosine residue 3-I-Tyr, 5-I-Tyr, wherein the iodine may be a radioactive isotope, e.g., 11251 127, or 1131. An aliphatic amino acid is an a-amino acid having one or two side chains which, independently, 20 are hydrocarbons, a straight or branched chain of 1-6 carbons. Examples of aliphatic amino acids include Ala, Aib, Val, Leu, Ile, Nle, Nva, or Abu. An aromatic *amino acid is an a-amino acid the side chain of which has a neutral not acidic or basic) aromatic substituent, a substituted or unsubstituted phenyl, naphthyl, or aromatic heterocycle group pyridyl or indolyl). Examples of aromatic amino acids include Phe, p-X-Phe (where X is a halogen F, Cl, or OH,

OCH

3

CH

3 or NO 2 o-X-Phe (where X is a halogen, OH,

OCH

3

CH

3 or NO 2 pyridyl-Ala, Trp, P-Nal, 2,4-dichloro- Phe, Tyr(I), F 5 -Phe. Where the amino acid residue is optically active, it is the L-isomer that is intended unless otherwise specified. Also, in the above generic formula, hydroxyalkyl, hydroxyphenylalkyl, and hydroxynaphthylalkyl may contain 1-4 hydroxy substituents, and COE 1 stands for -C=OE 1 Examples of H: \marar\wip\K-\P36702-div.doc 14/01/00 16 -C=O EI include, but are not limited to, p-hydroxyphenylpropionyl -C=0 CH 2

-CH

2

-C

6

H

4 -OH) and phenylpropionyl. In the formula set forth herein, the disulfide bond between the thiol group on the side chain of residue A 1 Mpa, D-Mpa, Cys, or D-Cys) and the thiol group on the side chain of residue Ag Mpa, D- Mpa, Cys, or D-Cys) is not shown. A peptide of this invention is also denoted herein by another format, e.g.,

H

2 -c[Cys-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys]-NH 2 with the two disulfide bonded residues Cys) placed between the two brackets following The peptides can be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those formed with organic acids acetic, lactic, maleic, citric, malic, 0 ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids hydrochloric acid, sulfuric acid, or phosphoric acid), 20 polymeric acids tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids).

S

A therapeutically effective amount of a peptide oo. 25 and a pharmaceutically acceptable carrier substance magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can H:%\marar\wip\K-O\P36702-div.doc 14/01/00 17 form a micelle) together form a therapeutic composition a pill, tablet, capsule, or liquid) for administration orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, iontophoretically, or by intratracheally) to a subject in need of the peptide. The pill, tablet, or capsule can be coated with a substance capable of protecting the composition from the gastric acid or intestinal enzymes in the subject's stomach for a period of time sufficient to allow the composition to pass undigested into the subject's small intestine. The therapeutic composition can also be in the form of a biodegradable or nonbiodegradable sustained release formulation for subcutaneous or intramuscular 15 administration. See, U.S. Patents 3,773,919 and 4,767,628 and PCT Application No. WO 94/00148.

Continuous administration can also be obtained using an Simplantable or external pump INFUSAID pump) to administer the therapeutic composition. The peptide can 20 be administered prior to bedtime of the patient.

The dose of a peptide for treating the abovementioned diseases or disorders varies depending upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the peptide as determined by the attending physician or veterinarian is referred to herein as a "therapeutically effective amount".

H: \marar\wip\K-O\P367O2-di-.doc 14/01/00 18 Other features and advantages of the present invention will be apparent from the detailed description and from the claims.

Detailed Description of the Invention The synthesis and use of somatostatin analogs of this invention are well within the ability of a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 1 in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

It is believed that one skilled in the art can, 15 based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Synthesis of Somatostatin Analogs The synthesis of short peptides is well examined in the peptide art. See Stewart, et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984). The following is the synthesis of Analog I.

Other peptides of the invention can be prepared in an analogous manner by a person of ordinary skill in the art.

Benzylhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville, KY) (1.1 g, 0.5 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced ChemTech ACT 200 peptide synthesizer programmed to deliver the following reagents/solvents: methylene chloride; 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each); methylene H:\marar\wip\K-\P367O2-div-doc 14/01/00 19 chloride; ethanol; methylene chloride; and (f) triethylamine in chloroform.

The neutralized resin was stirred with Boc-S-4methylbenzyl-Cys and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h and the resulting amino acid resin was then cycled through steps to (f) in the above wash program. The following amino acids mmole) were then coupled successively by the same procedure: Boc-Phe, Boc-O-benzyl-Thr, Boc-Nbenzyloxycarbonyl-Lys, Boc-D-Trp, Boc-Phe, Boc-Phe, and Boc-S-methylbenzyl-Cys. After washing and drying, the completed resin weighed 1.6 g.

The resin (1.6 g, 0.5 mmole) was then mixed with anisole (5 ml), dithiothreitol (100 mg), and anhydrous hydrogen fluoride (35 ml) at 0OC and stirred for 45 min.

15 Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen, and the free peptide precipitated and washed with ether. The crude peptide was then S"dissolved in 500 ml of 90% acetic acid to which was added a concentrated solution of I2/MeOH until a permanent brown color was observed. Excess 12 was removed by addition of ascorbic acid, and the solution evaporated to a small volume which was applied to a column (2.5 x cm) of SEPHADEX" G-25 which was eluted with 50% AcOH.

Fractions containing a major component by ultraviolet (UV) absorption and thin layer chromatography were then pooled, evaporated to a small volume, and applied to a column (1.5 x 70 cm) of VYDAC octadecylsilane silica (10-15 gm). This column was eluted with a linear gradient from 80 percent A and 20 percent B to 40 percent A and 60 percent B where A is 0.1% trifluoroacetic acid (TFA) in water and B is 79.9% acetonitrile, 20% water, and 0.1% TFA. Fractions were examined by thin layer chromatography (tlc) and analytical high performance liquid chromatography (hplc) and pooled to give maximum purity. Repeated lyophilization of the solution from H: \-arar\ip\K-O\P367O2-div.doc 14/01/00 20 water gave 95 mg of the product as a white, fluffy powder.

The product is found to be homogenous by hplc and tic. Amino acid analysis of an acid hydrolysate and matrix-assisted laser desorption mass spectrometry (MALDI MS) confirmed the composition of the cyclic octapeptide (MW calculated, 1077; MW found, 1080).

The following is the synthesis of Analog V.

Benzylhydrylamine-polystyrene resin (Advanced ChemTech, Inc.) (0.7 g, 0.25 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced ChemTech ACT 200 peptide synthesizer programmed to deliver the following reagents/solvents: methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each); methylene chloride; (d) 15 ethanol; methylene chloride; and triethylamine in chloroform.

The neutralized resin was stirred with Boc-Smethylbenzyl-Cys and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h and the resulting 20 amino acid resin was then cycled through steps to (g) o in the above wash program. The following amino acids mmole) were then coupled successively by the same procedure: Boc-Phe, Boc-O-benzyl-Thr, Boc-N- •benzyloxycarbonyl-Lys, Boc-D-Trp, Boc-Phe, Boc-Phe, Boc- Asn, and Boc-S-methylbenzyl-Cys. After washing and drying, the completed resin weighed 1.2 g. The peptide resin was subjected to HF cleavage and 12 cyclization as described above. Column purification, as described above, yielded 21 mg of the cyclic nonapeptide which was found to be homogeneous by hplc and tic. Amino acid analysis of an acid hydrolysate and MALDI MS confirmed the composition of the cyclic nonapeptide (MW calculated, 1192; found, 1192).

The synthesis of iodinated somatostatin analogs at the tyrosine residue the chloramine-T method) .is well documented and are within the ability of a person H:\-arar\wip\K-O\P36702-div.dOc 14/01/00 21 of ordinary skill in the art. See, Czernick, et al., J. Biol. Chem. 258:5525 (1993) and European Patent No. 389,180 Bl.

Somatostatin Receptor Binding Assay Human SSTR-2 Binding Assay: CHO-K1 (ovary, Chinese hamster) cells were obtained from the American Type Culture Collection (ATCC)(Rockville, MD) (ATCC No. CCL61) and were transfected with the human SSTR-2 cDNA, described in Yamada, et al., Proc. Natl. Acad. Sci. USA, 89:251-255 (1992) and also available from ATCC (ATCC No. 79046), using standard techniques known in the molecular biological art. See, Patel, et al., Biochem.

Biophys. Res. Commun. 198:605 (1994). Crude membranes were prepared by homogenizing the human SSTR-2 transfected CHO-K1 cells in 20 ml of ice-cold 50 mM Tris- HC1 (Buffer A) with a POLYTRON' homogenizer (Brinkmann :Instruments, Westbury, NY) at setting 6, for 15 sec.

Additional Buffer A was added to obtain a final volume of 20 40 ml, and the homogenate was centrifuged in a SORVAL SS-34 rotor (DuPont, Newtown, CT) at 39,000 g for 10 min at 0-4 0 C. The resulting supernatant was decanted and discarded. The pellet was rehomogenized in ice-cold Buffer A, diluted, and centrifuged as before. The final pellet was resuspended in the 10 mM Tris HC1 and held on ice for the receptor binding assay.

Aliquots of the membrane preparation were 9 incubated for 90 min at 25°C with 0.05 nM 1 2 5 I-Tyr]MK-678 (2000 Ci/mmol; c(N-methyl-Ala-Tyr(I125)-D-Trp-Lys-Val- Phe]) in 50 mM HEPES (pH 7.4) containing a test peptide at various concentrations 10 1 1 to 10 6 mg/ml bovine serum albumin (fraction V, Sigma Chemical Co., St. Louis, MO), MgCl 2 (5 mM), Trasylol (200 KIU/ml), bacitracin (0.02 mg/ml), and phenylmethyl-sulphonyl fluoride (0.02 mg/ml). The final assay volume was 0.3 ml. The incubations were terminated by rapid filtration H:\mrar\wip\K-OP36702-div.doc 14/01/00 22 through GF/C filters (pre-soaked in 0.3% polyethylenimine for 30 min) using a filtration manifold (Brandel, Gaithersburg, MD). Each tube and filter was then washed three times with 5 ml aliquots of ice-cold Buffer A.

Specific binding was defined as the total [1 25 I-Tyr]MK-678 bound minus that bound in the presence of 200 nM somatostatin-14.

The following test peptides were assayed: somatostatin-14, somatostatin-28, Analog I, Analog II, Analog III, Analog IV, and Analog V. The structure of Analogs I-V are shown above. The Ki values for the test peptides were calculated by using the following formula: Ki ICso/[l+(LC/LEC)] where IC 50 is the concentration of the test peptide required to inhibit 50 percent of the 15 specific binding of the radioligand 125 I-TyrJMK-678, LC is the concentration of the radioligand (0.05 nM), and LEC is the equilibrium dissociation constant of the radioligand (0.155 nM). The K i values calculated for the test peptides are shown in the column under the heading "SSTR-2" in Table I.

Human SSTR-5 Binding Assay S"CHO-K1 cells were transfected with the human SSTR-5 cDNA, described in Yamada, et al., Biochem Biophys. Res. Commun., 195-844 (1993) using standard techniques known in the molecular biological art. See, Patel, et al., Biochem. Biophys. Res. Comm. 198:605 (1994). Crude membranes were prepared by homogenization of the human SSTR-5 transfected CHO-K1 cells in 20 ml of ice-cold 50 mM Tris-HCl with a POLYTRON" homogenizer (setting 6, 15 sec). Buffer was added to obtain a final volume of 40 ml, and the homogenate was centrifuged in a SORVAL SS-34 rotor at 39,000 g for 10 min at 0-4 0 C. The resulting supernatant was decanted and discarded. The pellet was rehomogenized in ice-cold buffer, diluted, and H:\mrar\Wip\K-O\P36702-div.doc 14/01/00 23 centrifuged as before. The final pellet was resuspended in the 10 mM Tris HC1 and held on ice for the receptor binding assay.

Aliquots of the membrane preparation were incubated for 30 min at 30°C with 0.05 nM [125,- Tyrll]somatostatin-14 (2000 Ci/mmol; Amersham Corp., Arlington Heights, IL) in 50 mM HEPES (pH 7.4) containing a test peptide at various concentrations 10- 11 to 10-6 10 mg/ml bovine serum albumin (fraction MgCl 2 mM), Trasylol (200 KIU/ml), bacitracin (0.02 mg/ml), and phenylmethyl-sulphonyl fluoride (0.02 mg/ml). The final assay volume was 0.3 ml. The incubations were terminated by rapid filtration through GF/C filters (presoaked in 0.3% polyethylenimine for 30 min) using a Brandel filtration manifold. Each tube and filter was then washed three times with 5 ml aliquots of ice-cold buffer. Specific binding was defined as the total [125I- S"Tyr 1 1]somatostatin-14 bound minus that bound in the presence of 1000 nM of the somatostatin type-5 receptor ligand BIM-23052 (H 2 -D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-

NH

2 The K, values for the test peptides were calculated by using the following formula: IC 50 where ICso is the concentration of the test peptide required to inhibit 50 percent of the specific binding of the radioligand 125 -Tyrll]somatostatin-14, LC is the concentration of the radioligand (0.05 nM), and LEC is the equilibrium dissociation constant of the radioligand (0.16 nM). The K i values calculated for the test peptides are shown in the column under the heading "SSTRin Table I.

Table I also shows the respective ratios of the Ki's for the human SSTR-2 and the Ki's for the human SSTR- The peptides of the invention Analogs I-V) have ratios unexpectedly greater than one and, thus, are more selective for the SSTR-5 than for the SSTR-2.

H:\marar\wip\K-O\P36702-div.doc 14/01/00 24 0**e 55 *9 5 .0*eCe

C

TABLE 1 COMPOUND J BSTR-2 BBTR-5 Somatostatin-l 4 0.187 0.883 0.212 somnAtostatin-2 8 0.242 0.383 0.632 Analog I15. 0.376 40.2 Analog I1 13.0 2.63 4.94 Analog 111 14.7 1.21 12.1 Analog IV 19.3 0.928 20.8 Analog V 129 2.43 53.1 Analog VI 6.19 0.34 18.2 15 Analog VII 8.07 4.145 1.94 Analog VIII 4.78. 0.27 17.7 Analog IX 6.205 4.77 1.30 Analog X 15.0 0.744 20.1.

Analog Xi 34.83 28.04 1.24 Analog XII 59.20 1 5.78 1 10.2 Analog XIII 32.32 1 2.01 16.1 other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. other aspects, advantages, and modifications are within the claims.

H: \n aar\.ip\K-O\P367O2-div-doc 14/01/00 24a Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

S

r 0 *0 o *o ooooo 4 **1

Claims

1. A method of inhibiting the release of insulin in a subject in need thereof, which comprises administering to said subject an effective amount of a peptide of the formula: Ri Al-A 2 -A 3 -A 4 -D-Trp-Lys-A 7 -A 8 -A 9 -R 3 R2 in which AI is the D- or L-isomer of Cys; A 2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A 7 is Thr, Ser, or an aliphatic amino acid; 20 As is an aromatic amino acid; A 9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, C1- 12 alkyl, C7- 20 phenylalkyl, C 11 20 naphthylalkyl, Ci- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, C 11 -2 0 hydroxynaphthylalkyl, or COE 1 25 where El is Ci- 12 alkyl, C7- 20 phenylalkyl, Cl- 20 naphthylalkyl, CI- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, or C 11 20 hydroxynaphthylalkyl; and R 3 is NH 2 or NHYCH 2 Z where Y is a C1- 12 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and A 9 or a pharmaceutically acceptable salt thereof.

2. A method of treating type II diabetes in a subject in need thereof, which comprises administering to said subject an effective amount of a peptide of the formula: H:\marar\wip\K-O\P36702-div.doc 14/01/00 26 Al-A 2 -A 3 -A 4 -D-Trp-Lys-A 7 -A-A 9 -R 3 R in which A, is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A 7 is Thr, Ser, or an aliphatic amino acid; A 8 is an aromatic amino acid; A9 is the D- or L-isomer of Cys; *each of R, and R 2 is, independently, H, CI- 1 2 alkyl, C 7 20 phenylalkyl, Cll.. 20 naphthylalkyl, Cl- 1 2 hydroxyalkyl, C 7 20 hydroxyphenylalkyl, Cll.. 2 0 hydroxynaphthylalkyl, or COE, where El is Cl- 1 2 alkyl, C 7 2 0 phenylalkyl, Cll- 2 0 naphthylalkyl, Cl- 1 2 hydroxyalkyl, C 7 2 0 hydroxyphenylalkyl, or Cll.. 2 0 hydroxynaphthylalkyl; and R 3 is NH 2 or NH-YCH 2 'Z where Y is a Cl.. 1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a peptide of the formula: A 1 -A 2 -A 3 -A 4 -D-Trp-Lys -A 7 -A 8 -A 9 -R 3 R 2 H: \mrar\wip\K-O\P36702-div. doe 14/01/00 27 in which AI is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; A 9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, C1- 12 alkyl, C7- 20 phenylalkyl, C 11 2 0 naphthylalkyl, Ci- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, C 11 -2 0 hydroxynaphthylalkyl, or COE 1 where El is C 1 -1 2 alkyl, C7-2 0 phenylalkyl, C 11 20 o"o naphthylalkyl, C1-i2 hydroxyalkyl, C7-20 hydroxyphenylalkyl, or Cl-2 0 hydroxynaphthylalkyl; and R 3 is NH 2 or NH-YCH 2 -Z where Y is a Cl-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof.

4. A method of imaging cells in vivo containing somatostatin receptors comprising administering a peptide of the formula: R1 Al-A 2 -A 3 -A 4 -D-Trp-Lys-A7-As-A 9 -R 3 R2 in which A, is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; H:\-arar\wip\K-O\P36702-div.doc 14/01/00 28 A 9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, C1-1 2 alkyl, C7- 20 phenylalkyl, C11-2 0 naphthylalkyl, CI- 12 hydroxyalkyl, C 7 -2 0 hydroxyphenylalkyl, C 11 2 0 hydroxynaphthylalkyl, or COE 1 where El is Ci- 12 alkyl, C7- 20 phenylalkyl, C 11 -2 0 naphthylalkyl, C1- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, or C 11 -2 0 hydroxynaphthylalkyl; and R 3 is NH 2 or NH-Y-CH 2 Z where Y is a C1-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and A 9 or a pharmaceutically acceptable salt thereof, provided that at least one aromatic amino acid of the formula Tyr(I) is present in the peptide; to a subject and detecting the presence of said peptide in 15 said subject.

A method of imaging cells in vitro containing somatostatin receptors comprising administering a peptide of the formula: R, A 1 -A 2 -A 3 -A 4 -D-Trp-Lys-A 7 -As-A 9 -R 3 R2 in which Ai is the D- or L-isomer of Cys; A 2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A 7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; A 9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, C1- 12 alkyl, C7-20 phenylalkyl, C 11 -2 0 naphthylalkyl, C- 12 hydroxyalkyl, H:\marar\wip\K-O\P36702-div.doc 14/01/00 29 C 7 20 hydroxyphenylalkyl, Cll.. 20 hydroxynaphthylalkyl, or COE, where El is Cl- 1 2 alkyl, C7,.. 2 0 phenylalkyl, Cll.. 2 0 naphthylalkyl, Cl-. 1 2 hydroxyalkyl, 20 hydroxyphenylalkyl, or Cll.. 20 hydroxynaphthylalkyl; and R 3 is NH 2 or NH*Y*CH 2 *Z where Y is a Cl.. 12 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof, provided that at least one aromatic amino acid of the formula Tyr(I), wherein said iodine is radioactive iodine, 125 1, 12I or 131, is present in the peptide; to cells and detecting the presence of said peptide in said cells.

6. The use of a peptide of the formula: Al-A 2 -A 3 -A 4 -D-Trp-Lys-A,-A 8 -A 9 -R 3 R 0: in which A, is the D- or L-isomer of Cys; A 2 is Asn, Gln, an aliphatic amino acid, an aromatic amino acid or is deleted; *A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A7, is Thr, Ser, or an aliphatic amino acid; A 8 is an aromatic amino acid; A 9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, Cl.. 12 alkyl, C7-.20 phenylalkyl, C11...2o naphthylalkyl, Cl.. 12 hydroxyalkyl, C7-. 20 hydroxyphenylalkyl, Cll.. 20 hydroxynaphthylalkyl, or COE, where El is C 1 2 alkyl, C7-. 2 0 phenylalkyl, Cll.. 20 naphthylalkyl, Cl.. 1 2 hydroxyalkyl, C7-. 20 hydroxyphenylalkyl, or Cll-..20 hydroxynaphthylalkyl; and H:\mrar\WiP\K-0\P36702-div-doC 14/01/00 30 R 3 is NH 2 or NHYCH 2 Z where Y is a CI-1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and Ag; or a pharmaceutically acceptable salt thereof, for inhibiting the release of insulin.

7. The use of a peptide of the formula: RI Al-A 2 -A 3 -A 4 -D-Trp-Lys-A 7 -A 8 -A 9 -R 3 R2 in which 15 Ai is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; 20 A 7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; A 9 is the D- or L-isomer of Cys; each of Ri and R 2 is, independently, H, CI- 1 2 alkyl, C 7 -2 0 phenylalkyl, c 1 1- 2 0 naphthylalkyl, C 1 1 2 hydroxyalkyl, 25 C 7 -2 0 hydroxyphenylalkyl, C 1 1 -2 0 hydroxynaphthylalkyl, or COE 1 where El is C1-12 alkyl, C7- 20 phenylalkyl, C 11 -2 0 naphthylalkyl, C1- 12 hydroxyalkyl, C7- 20 hydroxyphenylalkyl, or C 11 20 hydroxynaphthylalkyl; and R 3 is NH 2 or NHYCH 2 Z where Y is a C1- 1 2 hydrocarbon moiety and Z is H, OH, C0 2 H, or CONH 2 and a disulfide bond links the side chains of residues Ai and A 9 or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the release of insulin. H:\arar\wip\K-O\P367O2-div.dc 14/01/00 31

8. A peptide of the formula: A 1 -A 2 -A 3 -A 4 -D-Trp-Lys -A 7 -As-A 9 -R 3 in which A, is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A 7 is Thr, Ser, or an aliphatic amino acid; A 8 is an aromatic amino acid; *A9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, Cl.. 12 alkyl, *C 7 20 phenylalkyl, Cll. 2 0 naphthylalkyl, Cl- 1 2 hydroxyalkyl, C 7 20 hydroxyphenylalkyl, Cll- 2 0 hydroxynaphthylalkyl, or COE, where El is Cl- 1 2 alkyl, C 7 20 phenylalkyl, Cll- 2 0 naphthylalkyl, Cl.. 12 hydroxyalkyl, C 7 2 0 hydroxyphenylalkyl, or Cll- 2 0 hydroxynaphthylalkyl; and R 3 is NH 2 or NH*Y*CH 2 'Z where Y is a Cl- 1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof when used to inhibit the release of insulin.

9. The use of peptide of the formula: R Al-A 2 -A 3 -A 4 -D-Trp-Lys -A 7 -A 8 -A 9 -R 3 R2 in which A 1 is the D- or L-isomer of Cys; H: \.aar\.iP\K-Q\P36702-div.doc 14/01/00 32 A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A 7 is Thr, Ser, or an aliphatic amino acid; As is an aromatic amino acid; A9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, Cl.. 12 alkyl, C 7 20 phenylalkyl, Cll.. 20 naphthylalkyl, Cl.. 12 hydroxyalkyl, C 7 20 hydroxyphenylalkyl, Cll-.. 20 hydroxynaphthylalkyl, or COE, where El is C 1 1 2 alkyl, C 7 2 0 phenylalkyl, Cll.. 2 0 naphthylalkyl, Cl.. 1 2 hydroxyalkyl, C 7 2 0 hydroxyphenylalkyl, Or Cl-2 hydroxynaphthylaikyl; and R 3 is NH 2 or NH-Y*CH 2 *Z where Y is a Cl.. 1 2 hydrocarbon :0::15 moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and A 9 or a pharmaceutically acceptable salt thereof, for the treatment of type II diabetes.

10. The use of a peptide of the formula: A 1 -A 2 -A 3 -A 4 -D-Trp-Lys-A 7 -A 8 -A 9 -R 3 R in which A, is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A 7 is Thr, Ser, or an aliphatic amino acid; A 8 is an aromatic amino acid; A9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, CI-. 12 alkyl, C 7 20 phenylalkyl, Cll.. 2 0 naphthylalkyl, Cl.. 12 hydroxyalkyl, H;\Marar\wip\K-O\P36702-divdoc 14/01/00 33 C 7 20 hydroxyphenylalkyl, Cll- 2 0 hydroxynaphthylalkyl, or COE, where El is Cl.. 12 alkyl, C 7 2 0 phenylalkyl, Cll- 2 0 naphthylalkyl, C 1 1 2 hydroxyalkyl, C7-. 20 hydroxyphenylalkyl, or Cll-. 20 hydroxynaphthylalkyl; and R 3 is NH 2 or NH-YCH 2 *Z where Y is a Cl- 1 2 hydrocarbon moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A, and Ag; or a pharmaceutically acceptable salt thereof, in the manufacture of a medicamnent for the treatment of type II diabetes.

11. A peptide of the formula: :5::15 Al-A 2 -A 3 -A 4 -D-Trp-LyS-A7-A 8 -A-R 3 in which A 1 is the D- or L-isomer of Cys; A 2 is Asn, Gin, an aliphatic amino acid, an aromatic amino acid or is deleted; *A 3 is an aromatic amino acid; A 4 is His or an aromatic amino acid; A7 is Thr, Ser, or an aliphatic amino acid; *A 8 is an aromatic amino acid; A9 is the D- or L-isomer of Cys; each of R, and R 2 is, independently, H, C 1 12 alkyl, C7.. 20 phenylalkyl, Cl-2 naphthylalkyl, Cl... 12 hydroxyalkyl, C 7 2 0 hydroxyphenylalkyl, Cll-.20 hydroxynaphthylalkyl, or COE, where El is CI.. 12 alkyl, C7.. 2 0 phenylalkyl, naphthylalkyl, Cl.. 1 2 hydroxyalkyl, C7.. 2 0 hydroxyphenylalkyl, or Cl-2 hydroxynaphthylalkyl; and R 3 is NH 2 or NH-Y*CH 2 'Z where Y is a Cl.. 12 hydrocarbon 3 5 moiety and Z is H, OH, CO 2 H, or CONH 2 and a disulfide bond links the side chains of residues A 1 and A 9 or a pharmaceutically acceptable salt thereof, H:\nmarar\wip\K-\P36702-divdOc 14/01/00 34 when used to treat type II diabetes.

12. A pharmaceutical composition as claimed in claim 3 when used to inhibit the release of insulin.

13. A pharmaceutical composition as claimed in claim 3 when used to treat type II diabetes. Dated this 14th day of January 2000 BIOMEASURE INCORPORATED THE ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and 15 Trade Mark Attorneys of Australia o.. *o ft ad« H: narar\wip\K-O\P36702-div.doc 14/01/00