WO1996032126A1

WO1996032126A1 - Analogs of growth hormone-releasing factor

Info

Publication number: WO1996032126A1
Application number: PCT/US1996/004582
Authority: WO
Inventors: David H. Coy; William Murphy
Original assignee: The Administrators Of The Tulane Educational Fund
Priority date: 1995-04-14
Filing date: 1996-04-03
Publication date: 1996-10-17
Also published as: JPH11503454A; MX9707904A; CA2218173A1; EP0820296A1; US5847066A; EP0820296A4; AU5441096A

Abstract

A peptide which is a variant of the human growth hormone-releasing factor. The peptide, containing 23-28 amino acid residues, differs from its native counterpart at least at positions 8, 9, 16, 18, 24, 25, 27 and 28 and is potent in stimulating the release of growth hormone.

Description

ANALOGS OF GROWTH HORMONE-RELEASING FACTOR

Background of the Invention

Growth hormone (GH) or somatotropin is a 191-amino acid peptide which is secreted by the anterior pituitary. Growth hormone itself does not promote growth directly, but acts by stimulating the production of growth factors, such as the somatomedins produced by the liver. The ultimate effects of growth hormone is to stimulate the growth of the skeleton, connective tissue, muscles, and viscera. Inadequate levels of growth hormone in children cause retardation of growth. It also causes retarded development of secondary sexual characteristics, impaired development of larynx, and hypoglycemia.

The production of growth hormone is under the control of both releasing and inhibitory factors secreted from the hypothalamus. The primary releasing influence is effected by growth hormone-releasing factor (GRF), which is produced primarily in the arcuate nucleus of the hypothalamus and transported to the pituitary by portal circulation.

Substantial efforts have been devoted to

development of synthetic GRF analogs with greater

efficacy than native GRF in stimulating release of growth hormone. For structures of human GRF (hGRF) and GRF's of other sources, see Wehrenberg, W.B., et al., Hormone Res., 24:82 (1986).

Summary of the Invention

The invention relates to peptides covered by the following generic formula:

in which

A_x is the D- or L- isomer of an amino acid selected from the group consisting of Tyr and His, or is deleted;

A₂ is Aib, or the D- or L- isomer of an amino acid selected from the group consisting of Ala, N-Me-Ala, and Arg;

A₈ is Ala, Aib, or Gly;

A₉ is Ala, Aib, or Gly;

A₁₀ is Phe or p-X-Phe where X is OH, CH₃, or a halogen (e.g., F, Cl, Br, or I);

A₁₂ is Lys or N⁶-X-Lys where X is C_1-6 alkyl, C_1-6 acyl, C_1-6 hydroxyalkyl, or C_2-6 hydroxyacyl;

A₁₅ is Ala, Aib, or Gly;

A₁₆ is Ala, Aib, or Gly;

A₁₈ is Ala, Aib, or Gly;

A₂₁ is Lys or N^ε-X-Lys where X is C₁-C₆ alkyl, C₁-C₆ acyl, C₁-C₆ hydroxyalkyl, or C₂-C₆ hydroxyacyl;

A₂₂ is Ala, Aib, Gly, Leu, lle, Val, Nle, Nva, or Abu;

A₂₄ is Ala, Aib, Gaba, Gly or His;

A₂₅ is Ala, Aib, Gaba, Gly, His, or is deleted;

A₂₆ is Ala, Aib, Gaba, Gly, His, or is deleted;

A₂₇ is Ala, Aib, Gly, Leu, lie, Val, Nle, Nva, Abu, Gaba, β-Ala, Ava, His, or is deleted;

A₂₈ is Aib, the D- or L- isomer of Ala, Gaba, His, or is deleted; each of R₁ and R₂ is, independently, H, C_1-12 alkyl, C_7-20 phenylalkyl, C_11-20 naphthylalkyl, C_1-12

hydroxyalkyl, C_7-20 hydroxyphenylalkyl, C_11-20

hydroxynaphthylalkyl, or COE₁ where E₁ is C_1-12 alkyl, C_7- ₂₀ phenylalkyl, C_11-20 naphthylalkyl, C_1-12 hydroxyalkyl, C_7-20 hydroxyphenylalkyl, or C_11-20 hydroxynaphthylalkyl; and

R₃ is OH, NH₂, C_1-12 alkoxy, or NH·Y·CH₂·Z where Y is a C_1-12 hydrocarbon moiety (divalent, e.g., straight or branched alkyl group) and Z is H, OH, CO₂H, or C0NH₂; or a pharmaceutically acceptable salt thereof.

Below are examples of the peptides of this invention as covered by the above formula:

With the exception of the N-terminal amino acid, Gaba, β-Ala, and Ava, all abbreviations (e.g., Ala or A₂) of amino acids in this disclosure stand for the structure of an α-amino acid residue -NH-CH(R)-CO-, wherein R is a side chain of an amino acid (e.g., CH₃ for Ala). Similar structures are intended for the non-α-amino acid residues Gaba, β-Ala, and Ava. On the other hand, for the N- teπαinal amino acid, the abbreviation stands for the structure of =N-CH(R)-CO-, wherein R is a side chain determinant of an amino acid. N-Me-Ala, Nle, Nva, Abu, Gaba, β-Ala, Ava, and Aib are respective abbreviations of the following α-amino acids: N-methyl-alanine,

norleucine, norvaline, α-aminobutyric acid, γ- aminobutyric acid (4-aminobutanoic acid), β-alanine (3- aminopropionic acid), 5-aminovaleric acid (5- aminopentanoic acid), and α-aminoisobutyric acid. N^ε-X- Lys stands for the amino acid Lys wherein a hydrogen of the epsilon amino group is replaced by X. 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, hydroxyacyl, hydroxyphenyl-alkyl, and

hydroxynaphthylalkyl may contain 1-4 hydroxy

substituents, and COE₁ stands for -C=O·E₁. Examples of -C=O· E₁ include, but are not limited to, p-hydroxy- phenylpropionyl (or Hpp, i.e., -C=O· CH₂-CH₂-C₆H₄-OH) and phenylpropionyl.

The peptides of the invention can be used to stimulate the release of growth hormone in a subject (a mammal such as a human patient) . Thus, the peptides are useful in the treatment of physiological conditions in which growth hormone is of benefit, e.g., those patients who lack adequate endogenous growth hormone production such as the elderly. The peptides of the invention can be used to stimulate linear growth in patients of short stature, e.g., growth hormone deficient children. Other uses include the stimulation of tissue growth (e.g., skeletal, cell, and organ growth) , protein metabolism, carbohydrate metabolism, lipid metabolism, mineral metabolism, and connective tissue metabolism, all of which lead to improved physical strength and well-being. The peptides of the invention can also be used in the treatment of catabolic states (e.g., recovery from infection, surgery, and malnutrition), the stimulation of immune function, and enhancement of natural sleep

patterns. The peptides of the invention can also be used in stimulating the growth of animals (e.g., livestock).

The peptides of this invention 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 (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic,

methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids) .

A therapeutically effective amount of a peptide of this invention and a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle) together form a therapeutic composition (e.g., a pill, tablet, capsule, or liquid) for administration (e.g., 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 administration. See, e.g., 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 impIantable or external pump (e.g. , INFUSAID* pump) to administer the therapeutic composition. The peptide can be administered prior to bedtime of the patient.

The dose of a peptide of the present invention for treating the above-mentioned 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."

Also contemplated within the scope of this invention is a peptide covered by the above generic formula for use in treating diseases or disorders

associated with growth hormone deficiencies. The GRF analogs of this invention do not possess the undesirable arginine and methionine residues at the C-terminus, thereby, both decreasing the cost of

synthesis and enhancing the stability of the peptides.

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 GRF 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 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, 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

The peptides of the invention can be prepared by standard solid phase synthesis. See, e.g., Stewart, J.M., et al., Solid Phase Synthesis (Pierce Chemical Co., 2d. ed. 1984). The following is a description of how Analog #3 was prepared. Other peptides of the invention can be prepared in an analogous manner by a person of ordinary skill in the art.

Benzyhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville, KY) (1.25 g, 0.5 mmole) in the chloride ion form is placed in the reaction vessel of an Advanced ChemTech peptide synthesizer (ACT 200) programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% diisopropyl-ethylamine in methylene chloride.

The neutralized resin was stirred with Boc-Ala and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for l h and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-Ala, Boc-Ala, Boc-Aib, Boc-Leu, Boc-Ala, Boc-Lys(2-Cl-Z), Boc- Arg(Tos), Boc-Ala, Boc-Aib, Boc-Leu, Boc-Ala, Boc-Ala, Boc-Leu, Boc-Val, Boc-Lys(2-Cl-Z), Boc-Arg(Tos), Boc- Tyr(diClBzl), Boc-Ala, Boc-Aib, Boc-Thr (Bzl), Boc-Phe, Boc-Ile, Boc-Ala, Boc-Asp, Boc-D-Ala, Boc-Tyr (diClBzl). After removal of the last Boc group and washing (MeOH) and drying in air at ambient temperature, the completed resin weighed 2.5 g.

The completed resin (2.5 g, 0.5 mmole) was mixed with anisole (5 ml), dithiothreitol (100 mg) and

anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 45 min. Excess hydrogen fluoride is evaporated rapidly under a stream of dry nitrogen. The free peptide precipitated and was washed with ether. The crude peptide was then dissolved in a minimum volume of 2 M acetic acid and eluted on a column (2.5 x 100 cm) of SEPHADEX® G-50 (Phairmacia, Piscataway, NJ) using the same solvent. Fractions containing a major component,

determined by UV absorption and thin layer

chromatography, were then pooled, evaporated to a small volume, applied to a column (2.5 x 50 cm) of VYDAC" octadecylsilane silica (10-15 μm) (Separations Group, Hesperia, CA), and eluted with a linear gradient of 10- 45% acetonitrile in 0.1% trifluoroacetic acid in water.

The collected fractions were examined by thin layer chromatography and analytical high performance liquid chromatography and pooled to give maximum purity. Repeated lyophilization of the solution from water gave a product of a white, fluffy powder.

The product is found to be homogeneous by high performance gas chromatography (HPLC) and thin layer chromatography (TLC). Amino acid analysis of an acid hydrolysate confirms the composition of the octapeptide. Laser desorption MS gave a molecular weight of 2793 in agreement with the calculated value.

The peptide of the invention can also be prepared by fragment condensation methodology wherein fragments of the peptide are synthesized separately and subsequently coupled. This methodology generally results in a

significantly higher synthesis yield. Preferably, the amino acid of the fragment coupled to resin is not optically active (e.g., Aib or Gly), thereby eliminating the possibility of racemization upon cleavage from the resin. The following is a description of the synthesis of Analog #2 by fragment condensation methodology. Other peptides of the invention can be prepared in an analogous manner by a person of ordinary skill in the art.

Boc-Aib-O-CH₃-polystyrene-divinylbenzene copolymer (Merrifield) resin (2.5 g, 1.0 mmole) was placed in the reaction vessel of an Advanced ChemTech (ACT 200) peptide synthesizer programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% diisopropylethylamine in methylene chloride. The neutralized resin is stirred with Boc-N^G- tosyl-Arg and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. The following amino acids (1.5 mmole) were then coupled successively by the same

procedure: Boc-Leu, Boc-Ala, Boc-Lys(Z), Boc-Arg(Tos), and Boc-Ala.

The completed resin weighed 3.4 g and was

suspended in tetrahydrofuran (THF) (50 ml) and saturated K₂CO₃ (20 ml). Tetrabutylammonium hydrogensulfate (2.8 g) was then added and the mixture stirred at 50°C (18 h). THF was removed under vacuum and the remaining solution neutralized with solid KHSO₄ whereupon a buff colored oil precipitates. This oil was extracted into n-BuOH and repeatedly washed with water and evaporated to give Boc- Ala-Arg(Tos)-Lys(Z--Ala-Leu-Aib-OH as a white powder (1.08 g, 0.96 mmole, 96%). The material gave one spot upon TLC on SiO₂ plates (CHCl₃:MeOH:H₂O:45:10:1).

Boc-Ala-O-CH₃-polystyrene-divinylbenzene copolymer (Merrifield) resin (1.0 g, 0.5 mmole) was placed in the reaction vessel of an Advanced ChemTech peptide

synthesizer programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% diisopropylethylamine in methylene chloride.

The neutralized resin was stirred with Boc-Ala and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. Boc-Ala (1.5 mmoles) was then coupled and the resin subjected to the same cycle of events. The

fragment Boc-Ala-Arg(Tos)-Lys(Z)-Ala-L)u-Aib-OH (750 mg, 0.74 mmole), synthesized above, was then coupled to this resin in the presence of HBTU (170 mg), HOBt (170 mg), and 10% diisopropylethylamine/DMF (3 ml). The following amino acids (1.5 mmole) are then coupled successively in the presence of diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h: Boc-Aib, Boc-Leu, Boc- Ala, Boc-Ala, Boc-Leu, Boc-Val, Boc-Lys(2-Cl-Z), Boc- Arg(Tos), Boc-Tyr(diClBzl), Boc-Ala, Boc-Aib, Boc- Thr(Bzl), Boc-Phe, Boc-Ile, Boc-Ala, Boc-Asp(FMOC), Boc- D-Ala, Boc-Tyr (diClBzl). The completed resin weighed 1.95 g. The completed resin (0.96 g, 0.25 mmole) was stirred in 20 ml of a 50:50 mixture of dimethylformamide and n-butylamine (18 h). The reaction mixture was evaporated to an oil to which water was added to give a white powder (0.5 g).

This powder was subjected to cleavage with hydrogen fluoride and column purification as described above. Repeated lyophilization of the solution from water gave the desired product as a white, fluffy powder. The product was found to be homogeneous by HPLC and TLC. Amino acid analysis of an acid hydrolysate confirms the composition of the octapeptide. Laser desorption MS gave a MW of 2736 in agreement with the calculated value.

The substituents R₁ and R₂ of the above generic formula may be attached to the free amine of the N- terminal amino acid by standard methods known in the art. For example, alkyl groups, e.g., C_1-12 alkyl, may be attached using reductive alkylation. Hydroxyalkyl groups, e.g., C_1-12 hydroxyalkyl, may also be attached using reductive alkylation wherein the free hydroxy group is protected with a t-butyl ester. Acyl groups, e.g., COE₁, may be attached by coupling the free acid, e.g., E₁COOH, to the free amine of the N-terminal amino acid by mixing the completed resin with 3 molar equivalents of both the free acid and diisopropylcarbodiimide in methylene chloride for one hour and cycling the resulting resin through steps (a) to (f) in the above wash program. If the free acid contains a free hydroxy group, e.g., p- hydroxyphenylpropionic acid, then the coupling should be performed with an additional 3 molar equivalents of HOBT.

The full names for the abbreviations used in the above description of synthesis are as follows: Boc for t-butyloxycarbonyl, Tos for tosyl, N^G-Tosyl for tosyl at guanidyl site, Z for benzyloxycarbonyl, Bzl for benzyl, HBTU for (1H-benzotriazol-1-yl)-1,1,3,3,- tetramethyluronium hexafluorophosphate, HOBT for

hydroxybenzotriazole, and FMOC for 9- fluorenylmethyloxycarbonyl.

Activities

Pituitaries from adult Charles River CD® male rats (Charles River Laboratories, Wilmington, MA) housed under controlled conditions (lights on from 0500-1900 h) were dispersed and cultured using aseptic technique by

modification of previously described methods. See

Hoefer, M.T., et al., Mol. Cell Endocrinol. 35:229

(1984); Ben-Jonathan, N., et al., Methods Enzymol.

103:249 (1983); and Heiman, M.L., et al., Endocrinology 116:410 (1985). Pituitaries were removed from

decapitated rats, sectioned, and then placed into a siliconized, liquid scintillation vial containing 2 ml 0.2% trypsin (Worthington Biochemicals, Freehold, NJ) in sterile-filtered Krebs-Ringer bicarbonate buffer

supplemented with 1% bovine serum albumin, 14 mM glucose, modified Eagle medium (MEM) vitamin solution and MEM amino acids (Gibco Laboratories, Grand Island, NY)

(KRBGA). All glassware was siliconized as described by Sayers, et al., Endocrinology 88:1063 (1971). The fragments were incubated in a water bath for 35 min. at 37°C with agitation. The vial contents then were poured into a scintillation vial containing 2 ml 0.1% DNase (Sigma Chemical Co., St. Louis, MO) in KRBGA and

incubated for 2 min. at 37ºC with agitation. After incubation the tissue was decanted into a 15 ml

centrifuge tube and allowed to settle. Medium was discarded, and pituitary sections were washed 3 times with 1 ml fresh KRBGA. The cells were then dispersed in 2 ml 0.05% LBI (lima bean trypsin inhibitor, Worthing Biochemicals) by gently drawing the fragments into and expelling them out of a siliconized, fire-polished

Pasteur pipette. Dispersed cells were then filtered through a 630 μm diameter Nylon mesh (Tetko, Elmsford, NY) into a fresh 15 ml centrifuge tube. The first tube was rinsed with an additional 2 ml LBI which was also transferred to the second tube with filtering.

The dispersed cells were then further diluted with approximately 15 ml sterile-filtered Dulbecco's modified Eagle medium (Gibco), which was supplemented with 2.5% fetal calf serum (Gibco), 3% horse serum (Gibco), 10% fresh rat serum (stored on ice for no longer than 1 h) from the pituitary donors, 1% MEM nonessential amino acids (Gibco), gentamicin (10 ng/ml; Sigma) and nystatin (10,000 U/ml; Gibco). The cells were poured into a 50 ml round-bottomed glass extraction flask with a large diameter opening. Cells were counted with a

hemacytometer (approximately 2,000,000 cells per

pituitary) and randomly plated at a density of about 200,000 cells per well (Co-star cluster 24; Rochester Scientific Co., Rochester, NY). The plated cells were maintained in the above Dulbecco's medium in a humidified atmosphere of 95% air and 5% CO₂ at 37°C for 96 h.

In preparation for a hormone challenge, the cells were washed 3 times with medium 199 (Gibco) to remove old medium and floating cells. Each dose of a test compound (diluted in siliconized test tubes) was tested in the presence of 0.1 nM somatostatin-14 in triplicate or quadruplicate wells in a total volume of 1 ml medium 199 containing 1% BSA (fraction V; Sigma Chemical Co.).

After 3 h at 37°C in an air/carbon dioxide atmosphere (95/5%) , the medium was removed and stored at -20°C until assayed for growth hormone content. Growth hormone content was determined by standard radioimmunoassay for rat growth hormone. The components for the

radioimmunoassay of rat GH including antiserum, GH for radioiodination, and GH reference preparation as well as procedure was obtained from the National Hormone and Pituitary Program (via Ogden Biosciences Corp.,

Rockville, MD) . The EC₅₀, the concentration of test compound required to simulate 50 percent of maximal growth hormone release, for each test compound was calculated and normalized to the EC₅₀ value of hGRF(l- 29)NH₂, which was simultaneously tested in each assay as a reference standard to insure the uniformity of the results, utilizing the following formula: Normalized EC₅₀ of Test Compound =

EC₅₀ of hGRF(1-29)NH₂/EC₅₀ of Test Compound

The GH release potency of the standard, hGRF(1-26)NH₂, and ten test compounds of the invention are listed in Table I. The relative GH release potency was calculated by the following formula:

Relative GH Release Potency =

Normalized EC₅₀ of hGRF(1-26) NH₂/Normalized EC₅₀ of Test Compound

As the data in Table I indicates, GRF analogs of the present invention are more potent than hGRF(1-26)NH₂.

For example, Analog #1, Analog #2, and Analog #8, all of which contain 26 amino acid residues, are, respectively, 400, 420, and 130 times more potent than the standard peptide hGRF(1-26)NH₂, which also contains 26 amino acid residues.

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.

Claims

What is claimed is:

1. A peptide of the formula:

in which

A₁ is the D- or L- isomer of an amino acid selected from the group consisting of Tyr and His, or is deleted;

A₈ is Ala, Aib, or Gly;

A_g is Ala, Aib, or Gly;

A₁₀ is Phe or p-X-Phe where X is OH, CH₃, or a halogen;

A₁₂ is Lys or N^ε-X-Lys where X is C_1-6 alkyl, C_1-6 acyl, C_1-6 hydroxyalkyl, or C_2-6 hydroxyacyl;

A₁₅ is Ala, Aib, or Gly;

A₁₆ is Ala, Aib, or Gly;

A₁₈ is Ala, Aib, or Gly;

A₂₁ is Lys or N^ε-X-Lys where X is C₁-C₈ alkyl, C₁- C₆ acyl, C₁-C₆ hydroxyalkyl, or C₂-C₆ hydroxyacyl;

A₂₂ is Ala, Aib, Gly, Leu, lle, Val, Nle, Nva, or Abu;

A₂₄ is Ala, Aib, Gaba, Gly or His;

A₂₅ is Ala, Aib, Gaba, Gly, His or is deleted;

A₂₆ is Ala, Aib, Gaba, Gly, His or is deleted;

A₂₇ is Ala, Aib, Gly, Leu, lle, Val, Nle, Nva, Abu, Gaba, β-Ala, Ava, His, or is deleted;

A₂₈ is Aib, the D- or L- isomer of Ala, Gaba, His, or is deleted; each of R₂ and R₂ is, independently, H, C_1-12 alkyl, C_7-20 phenylalkyl, C_11-20 napthylalkyl, C_1-12 hydroxyalkyl, C_7-20

hydroxyphenyl, C_11-20 hydroxynapthylalkyl, or COE₁ where E₁ is C_1-12 alkyl, C_7-20 phenylalkyl, C_lλ-20 napthylalkyl, C_1-12 hydroxyalkyl, C_7-20 hydroxyphenylalkyl, or C_11-20 hydroxynapthylaIky1; and

R₃ is OH, NH₂, C_1-12 alkoxy, or NH- Y- CH₂- Z where Y is a C_1-12 hydrocarbon moiety and Z is H, OH, CO₂H, or CONH₂; or a pharmaceutically acceptable salt thereof.

2. A peptide of claim 1, wherein A₂₅ is Ala, A₂₆ is Ala, A₂₇ is Ala, Aib, Leu, lle, Val, Nle, Nva, Gaba, or Abu, and A₂₈ is Ala.

3. A peptide of claim 2, wherein A₈ is Ala or Aib, A₉ is Ala, A₁₀ is Phe or Tyr, A₁₂ is Lys, A₁₅ is Ala,

A₁₆ is Ala, A₁₈ is Ala or Aib, A₂₁ is Lys, A₂₂ is Ala, A₂₄ is Ala or Aib, and A₂₇ is Ala, Leu, Gaba, or Nle.

4. A peptide of claim 3, wherein A₈ is Aib, A₁₈ is Aib, and A₂₄ is Aib.

5. A peptide of claim 4, wherein A₁ is Tyr, and A₂ is D-Ala or L-Ala.

6. A peptide of claim 4, wherein A₁ is deleted, A₂ is D-Ala or L-Ala, R₁ is H, and R₂ is COE₁.

7. A peptide of claim 3 , wherein R₃ is

NH·Y·CH₂·Z.

8. A peptide of claim 1, wherein A₂₈ is deleted.

9. A peptide of claim 8, wherein A₂₅ is Ala, A₂₆ is Ala, and A₂₇ is Ala, Aib, Leu, lie, Val, Nle, Nva, Gaba, or Abu.

10. A peptide of claim 9, wherein A₈ is Ala or Aib, A₉ is Ala, A₁₀ is Phe or Tyr, A₁₂ is Lys, A₁₅ is Ala, A₁₆ is Ala, A₁₈ is Ala or Aib, A₂₁ is Lys, A₂₂ is Ala, A₂₄ is Ala or Aib, and A₂₇ is Ala, Leu, Gaba, or Nle.

11. A peptide of claim 10, wherein A₈ is Aib, A₁₈ is Aib, and A₂₄ is Aib.

12. A peptide of claim 11, wherein A_x is Tyr, and A₂ is D-Ala or L-Ala.

13. A peptide of claim 11, wherein A_x is deleted, A₂ is D-Ala or L-Ala, R-_^ is H, and R₂ is COE-^

14. A peptide of claim 10, wherein R₃ is NH-Y-CH₂-Z.

15. A peptide of claim 1, wherein A₂₇ is deleted and A₂₈ is deleted.

16. A peptide of claim 15, wherein A₂₅ is Ala, and A₂₆ is Ala.

17. A peptide of claim 16, wherein A₈ is Ala or Aib, A₉ is Ala, A₁₀ is Phe or Tyr, A₁₂ is Lys, A₁₅ is Ala, A₁₆ is Ala, A₁₈ is Ala or Aib, A₂₁ is Lys, A₂₂ is Ala, and A₂₄ is Ala or Aib.

18. A peptide of claim 17, wherein A₈ is Aib, A₁₈ is Aib, and A₂₄ is Aib.

19. A peptide of claim 18, wherein A₁ is Tyr, and A₂ is D-Ala or L-Ala.

20. A peptide of claim 18, wherein A₁ is deleted, A₂ is D-Ala or L-Ala, R₁ is H, and R₂ is COE₁.

21. A peptide of claim 17, wherein R₃ is NH· Y· CH₂· Z.

22. A peptide of claim 1 of the formula: