WO2000010589A1

WO2000010589A1 - Somatostatin analogs

Info

Publication number: WO2000010589A1
Application number: PCT/US1999/019090
Authority: WO
Inventors: Jeffrey D. White
Original assignee: White Jeffrey D
Priority date: 1998-08-24
Filing date: 1999-08-23
Publication date: 2000-03-02
Also published as: WO2000010589A9; AU6382999A

Abstract

The present invention relates to somatostatin analogs which comprises a chemically substituted heptapeptide sequence having the cysteine groups in the 1 and 6 position being linked together to form a disulfide bridge in the monocyclic configuration. Chemical modifications are made at the free amino group of cysteine of the peptide (1). It has been demonstrated that the somatostatin analog peptide can be modified by the addition of isocyanates, isothiocyanates, acid chlorides, chloroformates and glycidyl ethers (epoxides) at the free amino group, at the terminal cysteine resulting in a measurable enhancement of the ability of the chemically modified compounds to bind somatostatin receptors.

Description

SOMATOSTATIN ANALOGS

BACKGROUND OF THE INVENTION

Somatostatin is a cyclic tetradecapeptide which inhibits release of several

pituitary and intestinal factors that regulate cell proliferation, cell motility, and/or

secretion including growth hormone, adrenocorticotropin hormone, prolactin, thyroid

stimulating hormone, insulin, glucagon, motilin, gastric inhibitory peptide, vasoactive

intestinal peptide, secretin, cholecystokinin, bombesin, gastrin releasing peptide, gastrin,

thyroid releasing hormone, pancreatic polypeptide, cytokines (e.g., interleukins,

interferons), growth factors (e.g., epidermal growth factor, nerve growth factor), and

vasoactive amines (e.g., serotonin). Several of these factors are implicated in regulation

of normal cell proliferation, as well as in tumor cell proliferation and metastasis.

Native somatostatin has a very short half life in vivo. A large number of novel

analogues have been prepared in order to enhance the duration of effect, biological

activity and the selectivity of this hormone. A variety of somatostatin peptide analogs

have been produced by elimination of amino acids that are not absolutely required for

activity and substitution of the native L-amino acids with the corresponding D-amino

acid isomers. Thus, some of these analogs are long acting, more potent receptor agonists

than native somatostatin, due in part to the resistance of D-amino acids to enzyme

degradation. For example, the synthetic somatostatin analog octreotide acetate, which

has the amino acid sequence D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol) is more potent than native somatostatin in inhibition of growth factor release. Bauer et al. U.S. Patent

No. 4,395,403.

SUMMARY OF THE INVENTION

The present invention provides novel chemically modified somatostatin analogs,

structural derivatives of native somatostatin which bind a somatostatin receptor. Analogs

include both antagonists and agonists of somatostatin activity.

More particularly, the present invention provides a somatostatin analog which

comprises a chemically substituted heptapeptide sequence having the cysteine groups in

the 1 and 6 position being linked together to form an disulfide bridge in the monocyclic

configuration. Chemical modifications are made at the free amino group of cysteine of

the peptide below

I 1

A- NH - Cys-Tyr-D-T -Lys-Thr-Cys-D-Tyr-NH₂ I

It has been demonstrated that the somatostatin analog peptide can be modified by

the addition of isocyanates, isothiocyanates, acid chlorides, chloroformates and glycidyl

ethers (epoxides) at the free amino group, at the terminal cysteine resulting in a

measurable enhancement of the ability of the chemically modified compounds to bind

somatostatin receptors.

The following synthetic reaction schemes are used to generate the chemically

modified peptides.

HN-peptide

+ HCI

+ HCI

CH₂-HN-peptide

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph which illustrates that peptide 3502 suppresses secretion of

growth hormone. Figure 2 is a graph which shows that orally administered peptide 3502 prevents

normal pulsatile secretion of growth hormone.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are cyclic heptapeptide analogs of somatostatin

having the general structure of Formula I

I 1

A-NH - Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr-NH₂

wherein A is

and RI is C1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, C=O, or SO₂ ; and X, and X₂ are independently, flourine, chlorine, bromine, iodine, C1-C4 alkyl,

NO₂ or

O

II CH₃-C — ^•

The chemical modifications of Formula I are generated at the free amino group of

the terminal cysteine moiety of the heptapeptide sequence.

Preferred compounds of this invention include compounds of Formula I:

wherein A is

Ri O

I II

HN— C

RI is C1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, CO, or SO₂ ; and

X_! and X₂ are independently, flourine, chlorine, bromine, iodine, C1-C4 alkyl,

NO₂ or

O

II CH₃-C — •

Another preferred compound of this invention of Formula I was the following

structure wherein A is

Ri O

I II

HN— C- R, is

Y is CH₂ and

X, is hydrogen.

Other preferred somatostatin analogs are compounds of Formula I

O OH

wherein A is H?C- ^■CH₂-

Rl is C1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, C=O, or SO₂ ; and

X_] and X₂ are independently, flourine, chlorine, bromine, iodine, C1-C4 alkyl, 0 II

CH₃-C ^•

Another preferred somatostatin analog of this invention is a compound of

Formula I

O f OH

wherein A is H₂C- ^"CH2~ and

R, is

X, is hydrogen, and

Y is a bond.

The invention features compounds, compositions and methods for the treatment

of diseases in mammals associated with increased production or secretion of any factor

or factors which can be regulated by somatostatin, including but not limited to growth

hormone, insulin, glucagon and pancreatic exocrine secretion.

The compounds can be administered in the dosages used for somatostatin or,

because of their greater potency, in smaller dosages. The compounds of the invention

can be used for the treatment of cancer, particularly growth hormone- or growth factor-

dependent cancer (e.g., bone, cartilage, pancreas, prostate, or breast), acromegaly and

related hypersecretroy endocrine states, or of bleeding ulcers and in those suffering from pancreatitis or diarrhea. The compounds can also be used in the management of diabetes

and to protect the liver of patients suffering from cirrhosis or hepatitis. The compounds

can also be used to treat Alzheimer's disease and as gastric cytoprotective compounds for

ulcer therapy. The compounds will also be useful in treating diabetes-related

retinopathy, nephropathy and vascular disease. The anti-cancer activity of the

compounds may be related to their ability to antagonize the actions of cancer-related

growth factors such as epidermal growth factor, insulin-like growth factor (IGF-1), or

vasoactive endothelial growth factor (VEGF).

The analogs can be made available in the form of pharmaceutically acceptable

salts or complexes. Examples of therapeutically acceptable acids for the formulation of

salts of the somatostatin analogs are inorganic acids, such as hydrochloric acid, sulfuric

acid, phosphoric acid, and the organic lactic, maleic, citric, succinic, benzoic, salicylic,

toluensulfonic acids. Complexes are compounds of Formula I formed by the addition of

organic salts or hydroxides such as Ca and Zn salts or the addition polymeric organic

materials, such as tannic acid or carboxymethyl cellulose.

In other preferred embodiments, a therapeutically effective amount of the

somatostatin analog or pharmaceutically acceptable salt or complex thereof are combined

with a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose,

a phospholipid or mannitol) to form a pharmaceutical composition. Examples of

methods of administration of the therapeutic reagent of the pharmaceutical composition

thereof include a pill, tablet, capsule or liquid for oral administration. The

pharmaceutical composition can also be administered as an ointment, gel, cream or lotion for application to the skin, or as a solution capable of being administered intravenously,

parenterally, subcutaneously, transmucosally, intranasally or intraperitoneally in an

appropriate buffer if necessary. The solid forms of this therapeutic composition can be

coated with a substance capable of protecting the modified peptide from digestion by

gastric acid in the stomach for a period of time sufficient to allow the composition to

pass undisintegrated into the small intestine. The therapeutic composition can be

administered via a sustained release formulation or a dermal patch. The descriptions are

provided as examples and are not meant to limit the possibilities of therapeutic

compositions or methods of administration of the somatostatin analogs.

Examples

I 1

The cyclic heptapeptide NH₂ Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr-NH_{2 was}

purchased from Polypeptide Laboratories. The reagents used to modify the heptapeptide

are widely available from commercial sources.

In accordance with the present invention, numerous modified peptides have been

synthesized according to the synthetic schemes outlined below.

1) Reactions of isocyanate with the heptapeptide of Formula I:

Ri O Ri O

_N=c + H₂N-peptide ► _{HN c}__HN__pep_ticie

The heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was

suspended in anhydrous acetonitrile to yield a 1 mM concentration. Fifty to 100 μl of this mixture was placed in a microcentrifuge tube. One equivalent of triethylamine (100

mM in acetonitrile) was added with mixing, followed by the addition of 1 equivalent of

the isocyanate (100 mM in acetonitrile). The reactions with the isocyanates were

incubated at room temperature for 60 to 120 min. Ten μl of water was added. The

solvent was removed by evaporation under vacuum. The chemically modified peptides

were than purified by reverse phase HPLC.

2) Reactions with isothiocyanates:

Ri S Ri

N=c + H₂N-peptide ^► _HN_c— _HN-peptide

The heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was

suspended in anhydrous acetonitrite to yield a 1 mM concentration. Fifty to 100 μl of

this mixture was placed in a microcentrifuge tube. One equivalent of triethylamine (100

mM in a acetonitrile) was added with mixing, followed by the addition of one equivalent

of isocyanate (100 mM in acetonitrile). The reactions with the isothiocyanates were

incubated at room temperature for 18 hrs. The solvent was removed by evaporation

under vacuum. The chemically modified peptides were then purified by reverse phase

HPLC.

3) Reactions with acid chlorides:

HN-peptide + HCI

The heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was

suspended in anhydrous acetonitrile to yield a 1 mM concentration. Fifty to 100 μl of

this mixture was placed in a microcentrifuge tube. Two equivalents of triethylamine

(100 mM in acetonitrile) was added with mixing, followed by the addition of 1

equivalent of an acid chloride (100 mM in acetonitrile). The reactions with the acid

chlorides were incubated at room temperature for 60-120 min. The solvent was removed

by evaporation under vacuum. The chemically modified peptides were then purified by

reverse phase HPLC.

4) Reactions with chloroformates:

+ HCI

The heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was

(100 mM in acetonitrile) was added with mixing, followed by the addition of 1

equivalent of a chloro formate (100 mM in acetonitrile). The reactions with the chloroformates were incubated at room temperature for 60-120 min. The solvent was

removed by evaporation under vacuum. The chemically modified peptides were then

purified by reverse phase HPLC.

5) Reactions with glycidyl ethers (epoxides):

CH₂-HN-peptide

The heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was

suspended in anhydrous rhethanol to yield a 1 mM concentration. Fifty to 100 μl of this

mixture was placed in a microcentrifuge tube. One equivalent of triethylamine (100 mM

in acetonitrile), followed by the addition of 1 equivalent of a glycidyl ether (100 mM in

methanol). The reaction was incubated at 65° C for 6-8 hrs. The solvent was removed

by evaporation under vacuum. The chemically modified desired peptides were purified

by reverse phase HPLC.

In the resulting modified peptides, R is C1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, C=O, or SO₂ ; and

X, and X₂ are independently, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, NO₂ or

O

II

CH₃-C — ^■

HPLC purification of chemically modified amino acids. The dried reaction

product was resuspended in 15μl of 100% of trifluoroacetic acid or 30 μl of 50%

trifluoroacetic acid over a period of 5-10 minutes. The volume was brought to lOOμl

with 50% acetonitrile and the mixture was injected onto a C18 reverse phase HPLC

column. The desired product was eluted from the column in a linear gradient of 0.1%

trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile (B) progressing

from 5% B at initial conditions to 60% B over 40 minutes. The elution position of the

desired product was monitored by UV absorbance and the desired peak was collected by hand in polypropylene tubes as it eluted from the UV detector. The eluent was dried

under vacuum then used for binding and bioassays.

A variety of tissues and cancers express somatostatin receptors. Five human

somatostatin receptors (hsstl, hsst2, hsst3, hsst4, hsst5) have been identified and cloned.

(Patel, Y.C., Life Sciences, Vol. 57, No. 13, pp. 1249-1265, 1995). Expression of these

five receptor subtypes varies with tissue types. Somatostatin receptor subtype 2 is

expressed on a wide variety of tumor types.

Receptor Binding Assays

Cell culture. CHO-Kl cells were grown as monolayers in Dulbecco's Modified Eagle's medium (DMEM, Mediatech, Washington DC) supplemented with 10% fetal calf serum, non-essential amino acids, 2mM glutamine, lmM pyruvate and 500 mg/mL gentamycin in 5% CO2 at 37°C.

Expression of hsst in CHO-Kl cells. For binding studies, CHO-Kl cell lines stably expressing hsst were created and propagated. The predicted coding region of each hsst was generated by PCR from human genomic DNA and ohgonucleotides corresponding to the coding region 5' and 3' ends as primers. The DNA fragment generated by PCR contained a Hind III restriction site at the 5' end and a Not 1 restriction site at the 3' end. The fragment was digested with these two restriction enzymes and directionally subcloned into the Hind Ill/Not 1 sites of the mammalian expression vector pCDNAl. The identity of each insert was verified by DNA sequencing. The construct was co-transfected with pSV2neo into a CHO-Kl cell line using the calcium phosphate protocol. Stable transfectants were selected using 400 mg/mL G418 and maintained in supplemented DMEM. After an initial ligand binding screen, one stable clone for each sst was chosen for all subsequent experiments. Preparation of Plasma Membranes. CHO/sst cells grown on 100mm tissue culture dishes were washed with ice cold PBS then scraped into 5ml of 50mM HEPES, pH 7.4-5mM MgCl₂ - 200 KIU/mL aprotinin - 2mg/mL PMSF and 2 mg/mL bacitracin (homogenization buffer). After a 15 min. incubation at 4°C, the cells were homogenized on ice using a Brinkman Polytron (setting 5, 15 sec) then re-homogenized with a hand held homogenizer (6 strokes). After centrifugation at 500 x g for 5 min. at 4°C, the supernatant was centrifuged again at 12,000 x g for 25 minutes at 4°C. The final pellet was resuspended in homogenization buffer. Protein content was measured using the bicinchoninic acid protein assay using BSA as a standard.

Preparation of 96-well plates. Costar 96-well strip plates (cat. no. 9102) were coated with poly-1-lysine by incubating each well in 50μL lOOmg/mL poly-1-lysine for lhr at 22°C. Excess liquid was removed and the wells were air dried. Membranes (lOmg/well) were added in 20mM Hepes pH 7.6 followed by incubation overnight at 4°C to evaporate all liquid. Non-specific binding sites were blocked by incubating each well in 50μL homogenization buffer supplemented with 1% BSA (incubation buffer) for 30 min at RT. Binding assays were performed after removal of excess liquid.

[¹²⁵I-Tyr"] SS 14 binding. For receptor binding studies, membranes were incubated at

22°C with 0.03nM [¹²⁵I-Tyrⁿ] SS14 (obtained from Amersham) with or without test

compounds each at concentrations ranging from 10^"10M to 10^"6M in 50μL incubation

buffer. After a 1 hour incubation at 22°C, excess liquid was removed by gently tapping

plates onto absorbent filter paper. Membranes were washed twice with lOOμL ice cold

incubation buffer and radioactivity in each well was determined. Specific binding was

defined as the difference between the amount of [¹²⁵I-Tyr^H] SS14 bound in the absence and

presence of lμM unlabeled SS14. Ki was determined using software programs Ligand or

Prism. The purified peptides were tested for binding to one or more of the five human

somatostatin receptor subtypes. Table 1 lists the results of receptor binding studies for a

number of peptides assayed against the five human somatostatin receptor subtypes. The

data of Table 1 indicates the chemical modifications change the binding affinity of the

parent heptapeptide to the various receptor subtypes.

TABLE 1

Ki's Human Receptors (nM)

# Core Peptide Seq Reacting Compound sstl sst2 sst3 sst4 sst5

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyτ- none 48.00

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- 1-Adamentyl Isocyanate 2.6₅

NH₂ 3₅02 Cys-Tyr-D-T -Lys-Thr-Cys-D-Tyr- Benzyl Isocyanate 0.65 31 4 1566 3.5

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- 4-Chlorophenyl Isocyanate 2000 0.90 31.5 1411 6.32

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- 4-Methoxyphenyl Isocyanate 1 S 0.59 8.87 203 2.26

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- (R)-(+)-alpha-Methylbenzyl 1940 0.41 215 104 7.22 isocyanate

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- (S)-(-)-alpha-Methylbenzyl 522 0.81 21 361 4.25 isocyanate

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- (R)-(-)-l-(l-Naphthyl)ethyl isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- (S)-(+)-l-(l-Naphthyl)ethyl 147.00 isocyanate

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- 4-Nιtrophenyl isocyanate 604 0.84 234 959 4.19

NH₂

Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr- Phenyl isocyanate 778 0.44 121 769

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- glycidyl 2-methylphenyl ether

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 1000 0.33 1000

NH, Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 1 ,2 epoxy-3-phenoxypropene 148

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- [2,3 Epoxypropyl] benzene 36

NH,

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Chlorophenyl glycidyl ether 77

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-tert-Butyl phenyl glycidyl 12 ether

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- glycidyl 4-methoxyphenyl ether

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Benzenesulfonyl isocyanate 1 9

NH,

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Benzoyl isocyanate 3 4

NH,

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Benzoyl isothiocyanate 2 9 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Benzyl isothiocyanate 216 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Chlorobenzenesulfonyl 3 7 isocyanate NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- p-Toluenesulfonyl isocyanate 2 1 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Fluorophenyl isocyanate 1093 1 34 38 6 1664 10 1 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Phenethyl Isothiocyanate 103 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- p-Tolyl isocyanate 823 0 62 204 1120 636 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Tπfluoro-p-tolyl isocyanate 3 3 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Bromophenyl isocyanate 1206 1 05 31 5 1204 8 64 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 2-Phenylphenyl isocyanate 454 0 92 50 799 10 4 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Phenethyl isocyanate NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 2,6-Dιmethylphenyl isocyanate NH₂

Cys-Tyr-D-Ttp-Lys-Thr-Cys-D-Tyr- 4-Phenoxyphenyl isocyanate 1 9 NH₂ 3533 Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Acerylphenyl isocyanate 1522 0 21 29 9 1147 5 65 NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-n-Butylphenyl isocyanate 2 6 NH, Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Chloro-2-methylphenyl isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 2,4-Dιchlorobenzyl isocyanate 1.2 144 591

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 2,3-Dιmethylphenyl 0 69 84 3₅3 3 9 isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 2,4-Dιmethylphenyl 07 6 9 251 2 8 isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 2,5-Dιmethylphenyl 0.62 8 1 284 isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 3 ,4-Dιmethylpheny 1 077 11 7 105 3 9 isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 3 ,₅-Dιmethylphenyl 1334 043 10 7 561 3 03 isocyanate

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- 4-Ethylphenyl isocyanate 0 81 12 1 274 3 1

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- p-Toluoyl chloπde 1 35 17 6 340 1 7

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Cmnamoyl chloπde 1 01 19 3 432 4 5

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Phenyl chloroformate 2 58

NH₂

Cys-Tyr-D-Tφ-Lys-Thr-Cys-D-Tyr- Benzyl chloroformate

NH₂

Cys-Tyr-D-Tφ-Lys-Val-Cys-D-Tyr- 18

NH₂

Cys-Tyr-D-Tφ-Lys-Val-Cys-D-Tyr- benzyl isocyanate 1 8

NH₂

Cys-Tyr-D-Tφ-Lys-Val-Cys-D-Tyr- Phenyl isocyanate 4 1

NH₂

Cys-Tyr-D-Tφ-Lys-Val-Cys-D-Tyr- 4-Acetylphenyl isocyanate

NH,

Bioactivity Assays

The in vivo biological activity of modified peptides was determined by evaluating their inhibitory potency on pituitary growth hormone (GH) release in sodium pentobarbital-anesthetized rats. The pentobarbital treated rat is a well characterized and frequently used model for studying GH secretory dynamics (see K. Chihara, A. Armura and AV Schally 1979 Endocrinology 104 1434). Dose-Response Studies: Adult male Sprague-Dawley rats weighing 250-300g with jugular vein cannulas were obtained from Zivic-Miller Labs, Zelienople, PA. On the day of assay, the rats were anesthetized with sodium pentobarbital (60mg/kg of body weight, administered i.p.). Thirty minutes later, the animals were injected iv. with saline or test compound at doses ranging from 0.1 to 30 μg/kg. Blood samples (250 μL) were drawn from the jugular vein cannula 10 min prior to test compound injection (baseline) and 5, 15, 30, 45 and 60 minutes after injection. The plasma was separated and assayed for GH by RIA using material supplied by the National Hormone and Pituitary Program, and for glucagon and glucose using commercially available reagents. To prevent hemodynamic disturbances, the red blood cells were resuspended in normal saline and returned to the animal. Figure 1 illustrates the dose response of peptide 3502 at three dosage levels 5 μg/kg, 2.5 μg/kg, and 1 μg/kg. At each dosage, the peptide was shown to be effective in suppressing production of growth hormone.

Time-Course Assay: Groups of cannulated rats were treated with sodium pentobarbital as in the dose-response assay. Thirty minutes later animals were injected via the jugular cannula with saline or test compound at the minimum dose giving maximal GH inhibition. Sodium pentobarbital at half the initial dose was given at 60- to 90-minute intervals to maintain anesthesia. Blood (250 μL) was collected from the jugular vein at approximately 15, 30, 60, 120, 180, and 240 min. after the injection of test compound and treated as described above. Data from the time-course assay are shown in Table 2. The results of this experiment demonstrates that compounds 3502 and 3533 block the ability of arginine to stimulate glucagon secretion, mimicking a normal function of somatostatin. The alpha cells which secrete glucagon are known to express the SST₂ receptor; thus this activity of the peptides is consistent with their selectivity for the SST₂ receptor.

The data also show that the peptides do not cause hypoglycemia, either alone or in combination with arginine. Assay: Glucagon (pg/ml) GH Glucose (mg/dl)

(ng/ml)

Treatment mean sem mean sem mean sem

Time -35 min pentobarbital 70mg/kg -15 min 47.1 8.6 255.4 175.4 153.1 8.1 -10 min saline -5 min 40.6 4.5 64.5 32.3 140.4 8.8 0 min L-arg. ,400mg/kg 5min 102.1 7.2 43.9 18.4 160.3 10.9 10 min 82.8 7.7 28.4 10.9 162.0 14.4 15 min 63.7 0.9 26.5 10.8 136.7 13.8 30min 49.7 6.1 46.0 14.4 103.1 1 1.9 45 min 45.1 3.6 57.4 13.8 91.9 5.1 60 min 41.2 4.4 59.1 4.2 103.9 3.0 75 min 42.7 10.8 54.3 8.2 107.2 2.6

Treatment:

Time -35 min pentobarbital 70mg/kg -15 min 50.2 2.0 283.7 208.8 152.5 1.9 -10 min MS3502, 5ug/kg -5 min 43.1 8.3 72.1 31.2 143.7 3.2 0 min L-arg. ,400mg/kg 5min 76.2 8.8 24.3 8.1 162.4 4.0 10 min 62.8 12.6 12.7 3.5 150.3 8.3 15 min 38.9 18.0 9.7 2.4 128.4 7.7

30min 29.9 11.8 6.4 0.8 89.9 6.5 45 min 34.1 10.5 13.4 5.9 75.9 8.0 60 min 34.6 10.0 22.5 11.3 88.0 7.0 75 min 37.5 4.6 27.0 10.7 98.7 3.2

Treatment:

Time -35 min pentobarbital 70mg/kg -15 min 50.1 8.4 284.3 230.8 157.7 7.1 -10 min MS3533, 5ug/kg -5 min 40.4 7.5 79.0 46.9 150.0 2.3 0 min L-arg. ,400mg/kg 5min 73.2 11.6 34.8 23.5 162.2 8.7 10 min 65.2 10.2 28.1 18.0 156.5 1.2 15 min 46.1 4.0 20.3 10.5 138.6 6.0 30min 38.0 2.4 12.2 3.6 100.9 4.5 45 min 37.5 6.1 12.5 3.8 88.9 1.7 60 min 39.2 8.2 14.5 7.5 88.8 8.4 75 min 46.3 7.9 15.1 6.8 103.4 14.6

Treatment:

Time

-35 min pentobarbital 70π

-15 min 40.8 5.7 89.9 45.7 153.4 7.7

-10 min MS3502, 1ug/kg

-5 min 35.1 3.2 41 .9 16.4 145.7 0.9

0 min L-arg. ,400mg/kg

5min 95.6 11.6 26.1 9.9 161.6 5.9 10 min 72.2 4.2 21.2 5.7 162.4 14.0 15 min 50.9 3.7 18.8 5.7 126.8 8.8 30min 43.9 6.3 39.5 18.4 91.9 5.4 45 min 43.5 6.0 43.4 5.9 77.7 3.0 60 min 36.2 4.2 65.5 14.9 90.1 4.8 75 min 36.6 7.8 60.4 17.7 102.3 0.1

Treatment:

Time -35 min pentobarbital 70mg/kg -15 min 49.5 4.9 137.0 73.1 157.2 1.5 -10 min MS3533, 1 ug/kg -5 min 41.7 6.1 61.8 21.0 144.6 6.8 0 min L-arg. ,400mg/kg 5min 111.0 17.5 32.2 6.4 159.2 1.4 10 min 77.3 10.2 23.5 5.1 151.5 3.1 15 min 59.8 3.2 19.3 1.0 128.3 4.1 30min 48.8 4.4 40.6 13.7 93.5 6.9 45 min 47.9 5.7 53.1 21.8 89.6 1.6 60 min 39.6 8.4 45.3 14.3 99.2 9.0 75 min 33.8 17.0 24.7 12.4 76.0 38.2

Treatment:

Time -35 min pentobarbital 70mg/kg -15 min 57.0 3.76 -10 min MS3502, 5ug/kg 46.9 10.2 170.1 12.3

oδ

B O N N CM CM 00 CD I CD

Έ

1

CO

CO sz v> co

E c

CO O co

CO 10

(0 co to

CO co c O -^

Oral activity: Adult male Sprague-Dawley rats weighing 250-300g with jugular vein and

gastric cannulas were obtained from Zivic-Miller Labs, Zelienople, PA. On the evening

prior to assay, rats were given 5 gram of food to eat with free access to water. On the day

of assay, the rats were anesthetized with sodium pentobarbital (60mg/kg of body weight,

administered i.p.). Thirty minutes later, the animals were injected through the gastric

cannula with saline or test compound at doses ranging from 0.1 to 30 μg/kg in a total

volume of 200 μL. Sodium pentobarbital at half the initial dose was given at 60- to 90-

minute intervals to maintain anesthesia. Blood (250 μL) was collected from the jugular

vein at approximately 15, 30, 60, 120, 180, and 240 min. after the injection of test

compound and treated as described above. In Figure 2 the graph representing oral saline

illustrates the cyclic increase and decrease of growth hormone levels during normal

secretion. The graph representing peptide 3502 shows that the peptide, orally

administered, prevents the normal secretion of growth hormone.

Claims

WHAT IS CLAIMED IS:

A modified heptapeptide of Formula I

A-NH - Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr-NH₂

wherein A is

O ╬¿ OH

H₉C- -CH₂- and R_] is C1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, CO, or SO₂ ; and

X, and X₂ are independently hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl,

NO₂ or

O

II

CH₃-C ΓÇö

Ri O

I II

2. The modified heptapeptide of Claim 1 wherein A is ^{HN c"} and R, is C1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, C=O, or SO₂; and

O

II CH₃-C ^ΓÇó

3. The modified heptapeptide of Claim 2, wherein

R, is

Y is CH₂ and

X, is hydrogen.

4. The modified heptapeptide of Claim 1 , wherein A is

O v OH

I j H₂C CH ^ΓÇö

1-C4 alkyl, adamantyl,

Y is a bond, C1-C4 alkenyl, C=O, SO₂; and

X, and X₂ are independently, fluorine, chlorine, bromine, iodine, C1-C4 alkyl,

O

II CH₃-C ^Γûá

5. The modified heptapeptide of Claim 4 wherein

Y is a bond and X, is hydrogen.

6. A pharmaceutical composition comprising a compound of Formula I, or a

pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising a modified heptapeptide of

Claim 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable

carrier.

8. A pharmaceutical composition comprising a modified heptapeptide of

Claim 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable

carrier.

9. A method for inhibiting the release of growth hormone, insulin, and

glucagon in a mammal comprising administering to the mammal a modified heptapeptide

of Formula I.