Antagonistic analogs of GH-RH inhibiting IGF-I and -II Number:7,026,281 from the United States Patent and Trademark Office (PTO) owispatent

Title: Antagonistic analogs of GH-RH inhibiting IGF-I and -II

Abstract: There is provided a novel series of synthetic analogs of hGH-RH(1-29)NH₂. These analogs inhibit the activity of endogenous hGH-RH, and therefore prevent the release of growth hormone. The stronger inhibitory potencies of the new analogs, as compared to previously described ones, results from replacement of various amino acids.

Patent Number: 7,026,281 Issued on 04/11/2006 to Schally,   et al.

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Inventors:	Schally; Andrew V. (Metairie, LA); Varga; Jozsef (New Orleans, LA); Zarandi; Marta (Szeged, HU)
Assignee:	The Administrators of the Tulane Educational Fund (New Orleans, LA)
Appl. No.:	547215
Filed:	April 11, 2000

Current U.S. Class:	514/2; 530/324; 530/317
Current Intern'l Class:	A61K 38/00    (20060101)
Field of Search:	514/11,12,9,10,2 530/324,317

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References Cited [Referenced By]

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Foreign Patent Documents
WO-95/1670/7	Jun., 1995	WO.

Other References

Wang, J. et al. 2000 J. Biol. Chem. 275 (1): 507-513. Section 3.4 Counting, Permutations, and Combinations for counting rules. Seto et (1990, Biochem. Biophys. Res. Commun 167, 360-336). Bowie et al (Science, 1990, 247:1306-1310). Burgess et al (J of Cell Bio. 111:2129-2138, 1990). Lazar et al (Molecular and Cellular Biology, 1988, 8:1247-1252). Gura (Science, 1997, 278:1041-1042). Jain (Sci. Am., 1994, 271:58-65). Curti (Crit. Rev. in Oncology/Hematology, 1993, 14:29-39). Hartwell et al (Science, 1997, 278:1064-1068).

Primary Examiner: Yu; Misook
Attorney, Agent or Firm: Behr; Omri M.

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Goverment Interests

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FIELD OF INVENTION

This invention was made in part with Government support from the Medical Research Service of the Veterans Affairs Department. The Government has certain rights in this application.

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Parent Case Text

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RELATED APPLICATIONS

This application is a divisional application of application Ser. No. 09/199,381, filed Nov. 25, 1998 now U.S. Pat. No. 6,057,422 granted May 2, 2000.

The present invention relates to novel synthetic peptides which inhibit the release of growth hormone from the pituitary in mammals, and to therapeutic compositions containing these novel peptides.

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Claims

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What is claimed is:

1. A method of suppressing excessive levels of GH in a patient in need of same which comprises administering to said patient an effective amount of a peptide selected from the group having the formulae:

X—R¹—R²-Asp-Ala-R⁵—R⁶-Thr-R⁸—R⁹—R¹⁰-Arg-R¹²—R¹³—R¹⁴—R¹⁵—R¹⁶ Leu-R¹⁸—R¹⁹-Arg-R²¹—R²²-Leu-Gln-Asp-Ile-R²⁷—R²⁸—R²⁹—NH₂


wherein X is PhAc, IndAc, or Nac,

R¹ is Tyr or His,

R² is D-Ag [or D-Cit],

R⁵ is Ile or Val,

R⁶ is Phe, Nal or Phe(Y), in which Y=Cl,

R⁸ is Asn, Gln, Ala, or D-Asn,

R⁹ is Arg, Har, Lys, Om, D-Arg, D-Har, D-Lys, D-Om, Cit, Nle, Tyr (Me), Ser, Ala or Aib,

R¹⁰ is Tyr or or Tyr(Me),

R¹² is Lys,

R¹³ is Val or NIe,

R¹⁴ is Leu or Nle,

R¹⁵ is Gly, Ala, Abu, Nle or Gln,

R¹⁶ is Gln or Arg,

R¹⁸ is Ser or Nle,

R¹⁹ is Ala,

R²¹ is Lys,

R²² is Leu, Ala or Aib,

R²⁷ is Met, Leu, Nle, Abu, or D-Arg,

R²⁸ is Arg, D-Arg, or Ser,

R²⁹ is Arg, D-Arg, Har or D-Har,

provided that where R⁹ and R²⁸ are Ser, R²⁹ is other than Arg or Har, and pharmaceutically acceptable salts thereof.

2. A method of treating a patient having a cancer carrying receptors for IGF-I or -II which comprises administering to said patient an effective amount of a peptide selected from the group having the formulae:

X—R¹—R²-Asp-Ala-R⁵—R⁶-Thr-R⁸—R⁹—R¹⁰-Arg-R¹²—R¹³—R¹⁴—R¹⁵—R¹⁶-Leu- R¹⁸—R¹⁹-Arg-R²¹—R²²-Leu-Gln-Asp-Ile-R²⁷—R²⁸—R²⁹—NH₂


wherein X is PhAc, IndAc, or Nac,

R¹ is Tyr or His,

R² is D-Arg [or D-Cit],

R⁵ is Ile or Val,

R⁶ is Phe, Nal or Phe(Y), in which Y=Cl,

R⁸ is Asn, Gln, Ala, or D-Asn,

R⁹ is Arg, Har, Lys, Om, D-Arg, D-Har, D-Lys, D-Om, Cit, Nle, Tyr (Me), Ser, Ala or Aib,

R¹⁰ is Tyr or or Tyr(Me),

R¹² is Lys,

R¹³ is Val or Nle,

R¹⁴ is Leu or Nle,

R¹⁵ is Gly, Ala, Abu, Nle or Gln,

R¹⁶ is Gln or Arg,

R¹⁸ is Ser or Nle,

R¹⁹ is Ala,

R²¹ is Lys,

R²² is Leu, Ala or Aib,

R²⁷ is Met, Leu, Nie, Abu, or D-Arg,

R²⁸ is Arg, D-Arg, or Ser,

R²⁹ is Arg, D-Arg, Har or D-Har,

provided that where R⁹ and R²⁸ are Ser, R²⁹ is other than Arg or Har, and pharmaceutically acceptable salts thereof.

3. A method for inhibiting IGF-II levels in tumors (cancers) and the expression of mRNA for IGF-II in the same tumors in patients having such tumors, which comprises administering to said patient an effective amount a peptide selected from the group having the formulae:

X—R¹—R²-Ala-R⁵—R⁶-Thr-R⁸—R⁹—R¹⁰-Arg-R¹²—R¹³—R¹⁴—R¹⁵—R¹⁶-Leu-R18—R¹⁹-Arg-R²¹—R²²-Leu-Gln-Asp-Ile-R²⁷—R²⁸—R²⁹—NH₂


wherein X is PhAc, IndAc, or Nac,

R¹ is Tyr or His,

R² is D-Arg [or D-Cit],

R⁵ is Ile or Val,

R⁶ is Phe, NaI or Phe(Y), in which Y=Cl,

R⁸ is Asn, Gln, Ala, or D-Asn,

R⁹ is Arg, Har, Lys, Om, D-Arg, D-Har, D-Lys, D-Om, Cit, Nle, Tyr (Me), Ser, Ala or Aib,

R¹⁰ is Tyr or or Tyr(Me),

R¹² is Lys,

R¹³ is Val or Nle,

R¹⁴ is Leu or Nle,

R¹⁵ is Gly, Ala, Abu, Nle or Gln,

R¹⁶ is Gln or Arg,

R¹⁸ is Ser or Nle,

R¹⁹ is Ala,

R²¹ is Lys,

R²² is Leu, Ala or Aib,

R²⁷ is Met, Leu, Nle, Abu, or D-Arg,

R²⁸ is Arg, D-Arg, or Ser,

R²⁹ is Arg, D-Arg, Har or D-Har,

provided that where R⁹ and R²⁸ are Ser, R²⁹ is other than Arg or Har, and pharmaceutically acceptable salts thereof.

4. The method of claim 1 which comprises administering a compound having the formula [PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 1.

5. The method of claim 1 which comprises administering a compound having the formula [PhAc⁰, D-Arg², Phe(pCl)⁶, Har⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 3.

6. The method of claim 2 which comprises administering a compound having the formula [PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 1.

7. The method of claim 2 which comprises administering a compound having the formula [PhAc⁰, D-Arg², Phe(pCl)⁶, Har⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 3.

8. The method of claim 3 which comprises administering a compound having the formula [PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 1.

9. The method of claim 3 which comprises administering a compound having the formula [PhAc⁰, D-Arg², Phe(pCl)⁶, Har⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 3.

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Description

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BACKGROUND OF THE INVENTION

Growth Hormone ("GH") is a peptide having 191 amino acids which stimulates the production of numerous different growth factors, e.g. insulin-like growth factor I (IGF-I) and so promotes growth of numerous tissues (skeleton, connective tissue, muscle and viscera) and physiological activities (raising nucleic acid and protein synthesis and lipolysis, but lowering urea secretion).

Release of GH is under the control of releasing and inhibiting factors secreted by the hypothalamus. The primary releasing factor is growth hormone releasing hormone ("GH-RH"); human growth hormone-releasing hormone ("hGH-RH") is a peptide having 44 amino acids. The novel peptides of the present invention relate to analogues of hGH-RH having only residues 1 through 29 ("hGH-RH(1-29)NH₂"), i.e., to analogues of the peptide which has the amino acid sequence:

• Tyr-Ala-Asp-Ala-Ile⁵-Phe-Thr-Asn-Ser-Tyr¹⁰-Arg-Lys-Val-Leu-Gly¹⁵-Gln-Leu-Ser-Ala-Arg²⁰-Lys-Leu-Leu-Gln-Asp²⁵-Ile-Met-Ser-Arg²⁹—NH₂ (SEQ ID NO: 1).

GH has been implicated in several diseases. One disease in which GH is involved is acromegaly, in which excessive levels of GH are present. The abnormally enlarged facial and extremity bones of this disease can be treated by administering a GH-RH antagonist.

Further diseases involving GH are diabetic retinopathy and diabetic nephropathy. The damage to the retina and kidneys respectively in these diseases, believed to be due to GH, results in blindness or reduction in kidney function. This damage can be prevented or slowed by administration of an effective GH-RH antagonist.

However, the main applications of GH-RH antagonists would be in the field of cancer (A. V. Schally et al, in Growth Hormone Secretagogues in Clinical Practice, eds. Bercu, B. B. & Walker, R. F., Dekker, New York, pp. 145-162, 1998). IGF-I and -II are potent mitogens for various cancers. By suppressing GH secretion, GH-RH antagonists decreases the synthesis of IGF-I in the liver and other tissues. GH-RH antagonists also reduce the autocrine and paracrine production of IGF-I and/or IGF-II by various tumors. In several experimental cancers, treatment with antagonists of GH-RH produces a reduction in IGF-I and -II, concomitant to inhibition of tumor growth.

In an effort to intervene in these disease and other conditions, some investigators have attempted to control GH levels by using somatostatin, one inhibitor of GH release. However, somatostatin, if administered alone, does not suppress GH or IGF-I levels to a desired degree. If administered in combination with a GH-RH antagonist, somatostatin would improve suppression of IGF-I levels much better.

Scientists have investigated various modifications of GH-RH to elucidate the relationship of the structure of GH-RH to its activity in an effort to provide synthetic congeners with improved agonistic or antagonistic properties. Thus, it was early established that GH-RH fragment comprising residues 1 to 29, or GH-RH(1-29), is the minimum sequence necessary for biological activity. This fragment retains 50% or more of the potency of native GH-RH.

The first described GH-RH antagonist, [Ac-Tyr¹,D-Arg²]hGH-RH(1-29)NH₂, which is generally termed as the "standard antagonist" in the literature, was found to prevent the activation of rat anterior pituitary adenylate cyclase by hGH-RH(1-29)NH₂. The same peptide blocked the action of GH-RH on its receptors in the pituitary and hypothalamus, and inhibited the pulsatile growth hormone secretion.

A considerable number of patents and articles in the open literature disclose analogs of GH-RH which either act as agonists of GH-RH (i.e. act to stimulate the release of GH) or as antagonists of GH-RH (i.e. act to inhibit the release of GH). Most of these peptides are derived from the GH-RH(1-29) peptide sequence, with specific structural modifications which account for their enhanced agonistic or antagonistic properties.

Thus, U.S. Pat. No. 4,659,693 discloses GH-RH antagonistic analogs which contain certain N,N′-dialkyl-omega-guanidino alpha-amino acyl residues in position 2 of the GH-RH(1-29) sequence.

Published application WO 91/16923 reviews earlier attempts to alter the secondary structure of hGH-RH by modifying its amino acid sequence. These earlier attempts include: replacing Tyr¹, Ala², Asp³ or ASn⁸ with their D-isomers; replacing Asn⁸ with L- or D-Ser, D-Arg, Asn, Thr, Gin or D-Lys; replacing Ser⁹ with Ala to enhance amphiphilicity of the region; and replacing Gly¹⁵ with Ala or Aib. When R² in the analogs is D-Arg, and R⁸, R⁹, and R¹⁵ are substituted as indicated above, antagonistic activity is said to result. These antagonistic peptides are said to be suitable for administration as pharmaceutical compositions to treat conditions associated with excessive levels of GH, e.g., acromegaly.

The antagonistic activity of the hGH-RH analogue "[Ser⁹-Ψ[CH₂—NH]-Tyr¹⁰]hGH-RH(1-29)" of U.S. Pat. No. 5,084,555 was said to result from the pseudopeptide bond (i.e., a peptide bond reduced to a [CH₂—NH] linkage) between the R⁹ and R¹⁰ residues. However, the antagonistic properties of [Ser⁹-∩[CH₂—NH]-Tyr¹⁰]hGH-RH(1-29) were said to be inferior to the standard antagonist, [N-Ac-Tyr¹, D-Arg²]GH-RH(1-29)—NH₂.

U.S. Pat. No. 5,550,212, and U.S. patent application Ser. No. 08/642,472, assigned to the same assignee as the present application, disclose analogs of hGH-RH(1-29)NH₂ said to have enhanced antagonistic properties and prolonged duration of action. These properties are believed to result from replacement of various amino acids and acylation with aromatic or nonpolar acids at the N-terminus of GH-RH(1-29)NH₂. It is noted that in the above U.S. patent and U.S. patent application, R⁹ is always Ser, R¹⁸ is Gln or an amino acid forming a lactam bridge (i.e. Glu), R²⁸ is Ser, Asn, Asp, Ala or Abu, and R²⁹ is Agm, Arg—NH₂, Arg—OH, Cit-NH₂, Cit-OH, Har-NH₂, Har-OH, or an amino acid forming a lactam bridge (i.e. Lys or Orn).

SUMMARY OF THE INVENTION

There is provided a novel series of synthetic analogs of hGH-RH(1-29)NH₂. These analogs inhibit the activity of endogenous hGH-RH, and therefore prevent the release of growth hormone. The stronger inhibitory potencies of the new analogs, as compared to previously described ones, results from replacement of various amino acids.

Specifically, the invention relates to peptides comprising the formulae:

X—R¹—R²-Asp-Ala-R⁵—R⁶-Thr-R⁸—R⁹—R¹⁰-Arg-R¹²—R¹³—R¹⁴—R¹⁵—R¹⁶-Leu- R¹⁸—R¹⁹-Arg-R²¹—R²²-Leu-Gin-Asp-Ile-R²⁷—R²⁸—R²⁹—NH₂

• wherein X is PhAc, IndAc, Ibu, Nac, 1- or 2-Npr, or Fpr,

R¹ is Tyr or His,

• R² is D-Arg or D-Cit,
• R⁵ is Ile or Val,
• R⁶ is Phe, Nal or Phe(Y), in which Y=F, Cl, Br,
• R⁸ is Asn, Gin, Ser, Thr, Ala, D-Asn, D-Gln, D-Ser, D-Thr, Abu, D-Abu, or Aib,
• R⁹ is Arg, Har, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle, Tyr (Me), Ser, Ala or Aib,
• R¹⁰ is Tyr or Phe(Y), in which Y=H, F, Cl, Br, or OCH₃,
• R¹² is Lys, D-Lys, or Orn,
• R¹³ is Val or Nle,
• R¹⁴ is Leu or Nle,
• R¹⁵ is Gly, Ala, Abu, Nle or Gin,
• R¹⁶ is Gin or Arg,
• R¹⁸ is Ser or Nle,
• R¹⁹ is Ala or Abu,
• R²¹ is Lys or Orn,
• R²² is Leu, Ala or Aib,
• R²⁷ is Met, Leu, Nle, Abu, or D-Arg,
• R²⁸ is Arg, D-Arg, Ser, Asn, Asp, Ala or Abu,
• R²⁹ is Arg, D-Arg, Har or D-Har,
  and pharmaceutically acceptable salts thereof.

Among the preferred embodiment are peptides wherein X is PhAc, IndAc or Nac, R¹ is Tyr or His, R² is D-Arg, R⁵ is Ile, R⁶ is Phe(pCl), R⁸ is Asn or Abu, R⁹ is Arg or Har, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle, or Tyr (Me), R¹⁰ is Tyr or Tyr(Me), R¹² is Lys, R¹³ is Val or Nle, R¹⁴ is Leu or Nle, R¹⁵ is Abu, Ala, or Nle, R¹⁶ is Gin or Arg, R¹⁸ is Ser or Nle, R¹⁹ is Ala or Abu, R²¹ is Lys, R²⁷ is Nle or D-Arg, R²⁸ is D-Arg, Arg, or Ser, R²⁹ is D-Arg, Har or D-Har.

It is noted that the amino acid residues from 30 through 44 of the native GH-RH molecule do not appear to be essential to activity; nor does their identity appear to be critical. Therefore, it appears that the addition of some or all of these further amino acid residues to the C-terminus of the hGH-RH(1-29)—NH₂ analogues of the present invention will not affect the efficacy of these analogues as GH-RH antagonists. If some or all of these amino acids were added to the C-terminus of the hGH-RH(1-29)—NH₂ analogues, the added amino acid residues could be the same as residues 30 through 44 in the native hGH-RH sequence or reasonable equivalents.

Synthetic Methods.

The synthetic peptides are synthesized by a suitable method such as by exclusive solid phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution phase synthesis.

When the analogues of this invention are synthesized by solid-phase method, the C-terminus residue (here, R²⁹) is appropriately linked (anchored) to an inert solid support (resin) while bearing protecting groups for its alpha amino group (and, where appropriate, for its side chain functional group). After completion of this step, the alpha amino protecting group is removed from the anchored amino acid residue and the next amino acid residue, R²⁸; is added having its alpha amino group (as well as any appropriate side chain functional group) suitably protected, and so forth. The N-terminus protecting groups are removed after each residue is added, but the side chain protecting groups are not yet removed. After all the desired amino acids have been linked in the proper sequence, the peptide is cleaved from the support and freed from all side chain protecting group(s) under conditions that are minimally destructive towards residues in the sequence. This is be followed by a careful purification and scrupulous characterization of the synthetic product, so as to ensure that the desired structure is indeed the one obtained.

It is particularly preferred to protect the alpha amino function of the amino acids during the coupling step with an acid or base sensitive protecting group. Such protecting groups should have the properties of being stable in the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain and without racemization of any of the chiral centers contained therein. Suitable alpha amino protecting groups are Boc and Fmoc.

Medical Applications.

The hGH-RH antagonist peptides, or salts of these peptides, may be formulated in pharmaceutical dosage forms containing effective amounts thereof and administered to humans or animal for therapeutic or diagnostic purposes. The peptides may be used to suppress GH levels and to treat conditions associated with excessive levels of GH, e.g., diabetic retinopathy and nephropathy, and acromegaly. Also provided are methods for treating these diseases by administration of a composition of the invention to an individual needing such treatment. The main uses of GH-RH antagonists are however, in the field of cancer, for example human cancers of the breast, lung, colon, brain, pancreas, and prostate where the receptors for IGF-I or IGF-II are present.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I is a plot of volume changes of MXT mouse mammary cancers during treatment with certain GH-RH antagonists against days of treatment.

FIG. II is a plot of volume changes of MDA-MB-468 human breast cancers in nude mice during treatment with certain GH-RH antagonists against days of treatment.

FIG. III is a plot of volume changes of HT-29 human colon cancers in nude mice during treatment with certain GH-RH antagonists against days of treatment.

FIG. IV is a plot of volume changes of U87MG human glioblastomas in nude mice during treatment with a GH-RH antagonist against days of treatment.

FIG. V is a plot of volume changes of PC-3 human prostate cancers in nude mice during treatment with certain GH-RH antagonists against days of treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Abbreviations

The nomenclature used to define the peptides is that specified by the IUPAC-IUB Commissioner on Biochemical Nomenclature wherein, in accordance with conventional representation, the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus appears to the right. The term "natural amino acid" as used herein means one of the common, naturally occurring L-amino acids found in naturally occurring proteins: Gly, Ala, Val, Leu, lie, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met Phe, Tyr, Pro, Trp and His. When the natural amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented herein unless otherwise expressly indicated.

Non-coded amino acids, or amino acid analogues, are also incorporated into the GH-RH antagonists. ("Non-coded" amino acids are those amino acids which are not among the approximately 20 natural amino acids found in naturally occurring peptides.) Among the non-coded amino acids or amino acid analogues which may be used in the hGH-RH antagonist peptides are the following: by Abu is meant alpha amino butyric acid, by Aib is meant alpha amino isobutyric acid, by Har is meant homoarginine, by NaI is meant 2-naphthyl-alanine, by Nle is meant norleucine, and by Orn is meant ornithine. When these non-coded amino acids, or amino acid analogues, have isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

Abbreviations used herein are:

	Abu	α-aminobutyric acid
	Ac	acetyl
	AcOH	acetic acid
	Ac2O	acetic anhydride
	Aib	α-aminoisobutyric acid
	Boc	tert.butyloxycarbonyl
	Bom	benzyloxymethyl
	2BrZ	2-bromo-benzyloxycarbonyl
	cHx	cyclohexyl
	Cit	citrulline (2-amino-5-ureidovaleric acid)
	2ClZ	2-chloro-benzyloxycarbonyl
	DCM	dichloromethane
	DIC	N,N′-diisopropylcarbodiimide
	DIEA	diisopropylethylamine
	DMF	dimethylformamide
	Fmoc	fluorenylmethyloxycarbonyl
	Fpr	3-phenylpropionyl
	GH	growth hormone
	GH-RH	GH releasing hormone
	Har	homoarginine
	HBTU	2-(1H-Benzotriazol-1-yl)-1,1,3,3-
		tetramethyluronium hexaflourophosphate
	hGH-RH	human GH-RH
	HOBt	1-hydroxybenzotriazole
	HPLC	high performance liquid chromatography
	Ibu	isobutyryl
	IndAc	indole-3-acetyl
	MBHA	para-methylbenzhydrylamine
	MeOH	methanol
	MeCN	acetonitrile
	Nac	1-naphthylacetyl
	Nal	2-naphthylalanine
	NMM	N-methylmorpholine
	Npr	naphthylpropionyl
	PAM	phenylacetamidomethyl
	Phe(pCl)	para-chloro-phenylalanine
	PhAc	phenylacetyl
	rGH-RH	rat GH-RH
	RP-HPLC	reversed phase HPLC
	TFA	trifluoroacetic acid
	Tos	para-toluenesulfonyl
	Tyr(Me)	tyrosine methylether
	Z	benzyloxycarbonyl

B. The GH-RH Analogs

The hGH-RH analogues of the present invention were designed to increase the affinities of the peptides to the receptor, to improve metabolic stability and to maximize the amphiphilic secondary structure of the molecules. Many of these analogues cause very effective and long lasting inhibition of the GH release stimulated by hGH-RH(1-29)NH₂ in vitro and in vivo.

The following embodiments are specially preferred as having remarkable bioactivity:

[PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 1

[Indac⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 2

[PhAc⁰, D-Arg², Phe(pCl)⁶, Har⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 3

[PhAc⁰, D-Arg², Phe(pCl)⁶, Har⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁸]hGH-RH(1-29)NH₂ Peptide 4

[Nac⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 5

[PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 6

[PhAc⁰, His¹, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁹, Har²⁹]hGH-RH(1-29)NH₂ Peptide 7

[Nac⁰, His¹, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁹, Har²⁹]hGH-RH(1-29)NH₂ Peptide 8

[PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 9

[PhAc⁰, D-Arg², Phe(pCl)⁶, Abu¹⁵, Arg¹⁶, Nle²⁷ D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 10

[PhAc⁰, D-Arg², Phe(pCl)⁶, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 11

[PhAc⁰, D-Arg², Phe(pCl)⁶, Nle⁹, Abu¹⁵, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 12

[PhAc⁰, D-Arg², Phe(pCl)⁶, Nle¹³, Nle¹⁴ Abu¹⁵ Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 13

[PhAc⁰, D-Arg², Phe(pCl)⁶, Nle¹⁵, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 14

[PhAc⁰, D-Arg², Phe(pCl)⁶, Abu¹⁵, Nle¹⁸, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 15

[PhAc⁰, D-Arg², Phe(pCl)₆, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 16

[PhAc⁰, D-Arg², Phe(pCl)⁶, Abu¹⁵, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 17

[PhAc⁰, D-Arg², Phe(pCl)⁶, D-Abu⁸, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 18

[PhAc⁰, D-Arg², Phe(pCl)⁶, Tyr(Me)¹⁰, Abu¹⁵, D-Arg²⁷, Arg²⁸, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 19

[PhAc⁰, D-Arg², Phe(pCl)⁶, Tyr(Me)⁹, Abu¹⁵, D-Arg²⁷, Arg²⁸, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 20

[PhAc⁰, D-Ar², Phe(pCl)⁶, Abu¹⁵, D-Arg²⁷, Arg²,D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 21

[PhAc⁰, D-Arg², Phe(pCl)⁶, Abu⁸, Tyr(Me)¹⁰, Abu¹⁵, D-Arg²⁷, Arg²⁸, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 22

[PhAc⁰, D-Arg², Phe(pCl)⁶, D-Abu⁸, Tyr(Me)¹⁰, Abu¹⁵, D-Arg²⁷, Arg²⁸, D-Arg²⁹]hGH-RH(1-29)NH₂ Peptide 23

[PhAc⁰, D-Arg², Phe(pCl)⁶, Lys⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 24

[PhAc⁰, D-Arg², Phe(pCl)⁶, Orn⁹, Abu¹⁶, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 25

[PhAc⁰, D-Arg², Phe(pCl)⁶, D-Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 26

[PhAc⁰, D-Arg², Phe(pCl)⁶, D-Har⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 27

[PhAc⁰, D-Arg², Phe(pCl)⁶, D-Lys⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 28

[PhAc⁰, D-Arg², Phe(pCl)⁶, D-Orn⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 29

[PhAc⁰, D-Arg², Phe(pCl)⁶, Cit⁹, Abu¹⁶, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 30

Six very preferred embodiments have the formulae:

PhAc⁰-Tyr¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)⁶-Thr⁷-Asn⁸-Arg⁹-Tyr¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu¹⁴-Abu¹⁵-Gln¹⁶-Leu¹⁷-Ser¹⁸-Ala¹⁹-Arg²⁰-Lys²¹-Leu²²-Leu²³-Gln²⁴-Asp²⁵-Ile²⁶-Nle²⁷-D-Arg²⁸-Har29< /sup>—NH₂  Peptide 1


IndAc⁰-Tyr¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)⁶-Thr⁷-Asn⁸-Arg⁹-Tyr¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu14-Abu¹⁵-Gln¹⁶-Leu¹⁷-Ser¹⁸-Ala¹⁹-Arg²⁰-Lys²¹-Leu²²-Leu²³-Gln²⁴-Asp²⁵-Ile²⁶-Nle²⁷-D-Arg²⁸-Har²⁹ —NH₂  Peptide 2


PhAc⁰-Tyr¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)⁶-Thr⁷-Asn⁸-Har⁹-Tyr(Me)¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu¹⁴ -Abu¹⁵-Gln¹⁶-Leu¹⁷-Ser¹⁸-Ala¹⁹-Arg²⁰-Lys²¹-Leu²²-Leu²³-Gln²⁴-Asp²⁵-Ile²⁶-Nle²⁷-D-Arg²⁸-Har²⁹—NH₂  Peptide 3


PhAc⁰-Tyr¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)⁶-Thr⁷-Asn⁸-Arg⁹-Tyr(Me)¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu¹⁴ -Abu¹⁵-Gln¹⁶-Leu¹⁷-Ser¹⁸-Ala¹⁹-Arg²⁰-Lys²¹-Leu²²-Leu²³-Gln²⁴-Asp²⁵-Ile²⁶-Nle²⁷-D-Arg²⁸-Har²⁹—NH₂  Peptide 6


PhAc⁰-His¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)⁶-Thr⁷-Asn⁸-Arg⁹-Tyr¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu14-Abu¹⁵-Gln¹⁶-Leu¹⁷-Ser¹⁸-Ala¹⁹-Arg²⁰-Lys²¹-Leu²²-Leu²³-Gln²⁴-Asp²⁵-Ile²⁶-Nle²⁷-D-Arg²⁸-Har29< /sup>—NH₂  Peptide 7


Nac⁰-His¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)-Thr⁷-Asn⁸-Arg⁹-Tyr¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu¹⁴-Abu¹⁵ -Gln¹⁶-Leu¹⁷-Ser¹⁸-Ala¹⁹-Arg²⁰-Lys²¹-Leu²²-Leu²³-Gln²⁴-Asp²⁵-Ile²⁶-Nle²⁷-D-Arg²⁸-Har²⁹—N H₂  Peptide 8


Under well-established convention, these may be abbreviated as follows:

[PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 1

[IndAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 2

[PhAc⁰, D-Arg², Phe(pCl)⁶, Har⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 3

[PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Tyr(Me)¹⁰, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 6

[PhAc⁰, His¹, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 7

[Nac⁰, His¹, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂ Peptide 8
C. Method of Preparation

1. Overview of Synthesis

The peptides are synthesized by suitable methods such as by exclusive solid phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution phase synthesis. For example, the techniques of exclusive solid-phase synthesis are set forth in the textbook "Solid Phase Peptide Synthesis", J. M. Stewart and J. D. Young, Pierce Chem. Company, Rockford, 111, 1984 (2nd. ed.), and M. Bodanszky, "Principles of Peptide Synthesis", Springer Verlag, 1984. The hGH-RH antagonist peptides are preferably prepared using solid phase, synthesis, such as that generally described by Merrifield, J. Am. Chem. Soc., 85 p. 2149 (1963), although other equivalent chemical syntheses known in the art can also be used as previously mentioned.

The synthesis is carried out with amino acids that are protected at their alpha amino group. Urethane type protecting groups (Boc or Fmoc) are preferably used for the protection of the alpha amino group. The preferred protecting group is Boc.

In solid phase synthesis, the N-alpha-protected amino acid moiety which forms the aminoacyl group of the final peptide at the C-terminus is attached to a polymeric resin support via a chemical link. After completion of the coupling reaction, the alpha amino protecting group is selectively removed to allow subsequent coupling reactions to take place at the amino-terminus, preferably with 50% TFA in DCM when the N-alpha-protecting group is Boc. The remaining amino acids with similarly Boc-protected alpha amino groups are coupled stepwise to the free amino group of the preceding amino acid on the resin to obtain the desired peptide sequence. Because the amino acid residues are coupled to the alpha amino group of the C-terminus residue, growth of the synthetic hGH-RH analogue peptides begins at the C terminus and progresses toward the N-terminus. When the desired sequence has been obtained, the peptide is acylated at the N-terminus, and it is removed from the support polymer.

Each protected amino acid is used in excess (2.5 or 3 equivalents) and the coupling reactions are usually carried out in DCM, DMF or mixtures thereof. The extent of completion of the coupling reaction is monitored at each stage by the ninhydrin reaction. In cases where incomplete coupling is determined, the coupling procedure is repeated, or a capping by acetylation of unreacted amino groups is carried out, before removal of the alpha amino protecting group prior to the coupling of the next amino acid.

A typical synthesis cycle is shown in Table I.

TABLE I

Protocol for a Typical Synthetic Cycle Using Boc-strategy

		Mixing
		Time
Step	Reagent	(min)
1. Deprotection	50% TFA in DCM	5 + 25
	DCM wash	1
	2-propanol wash	1
2. Neutralization	5% DIEA in DCM	1
	DCM wash	1
	MeOH wash	1
	5% DIEA in DCM	3
	MeOH wash	1
	DCM wash (3 times)	1—1
3. Coupling	3 equiv. Boc-amino acid in DCM
	or DMF + 3 equiv. DIC or the preformed
	HOBt ester of the Boc-amino acid	60
	MeOH wash	2
	DCM wash	2
4. Acetylation	Ac2O in DCM (30%)	10 + 20
(if appropriate)	MeOH wash (3 times)	2
	DCM wash (3 times)	2

After completion of the synthesis, the cleavage of the peptide from the resin can be effected using procedures well known in peptide chemistry.

Some of the amino acid residues of the peptides have side chain functional groups which are reactive with reagents used in coupling or deprotection. When such side chain groups are present, suitable protecting groups are joined to these functional groups to prevent undesirable chemical reactions occurring during the reactions used to form the peptides. The following general rules are followed in selecting a particular side chain protecting group: (a) the protecting group preferably retains its protecting properties and is not split off under coupling conditions, (b) the protecting group should be stable under conditions for removing the alpha amino protecting group at each step of the synthesis, (c) the side chain protecting group must be removable upon the completion of the synthesis of the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.

The reactive side chain functional groups are preferably protected as follows: benzyl for Thr and Ser; 2-bromo-benzyloxycarbonyl for Tyr; p-toluene-sulfonyl or nitro for Arg and Har; 2-chlorobenzyloxycarbonyl or fluorenylmethyloxycarbonyl for Lys, Orn; benzyloxymethyl for His; and cyclohexyl or fluorenylmethyl for Asp and Glu. The side chains of Asn and Gln are unprotected.

3. Stepwise Coupling of Amino Acid Residues to the Support Polymer

The hGH-RH antagonist peptides may be synthesized on a variety of support polymers, i.e. MBHA, Merrifield, PAM or Wang resins. When N-alpha-Boc protected amino acids are used for synthesis, the preferred resin is MBHA. In this case, peptides with an amidated C-terminus are obtained upon cleavage from the support phase.

First, the C-terminal amino acid is attached to the neutralized MBHA resin, and then the subsequent amino acid couplings are carried out. Each protected amino acid is coupled in about a three-fold molar excess, with respect to resin-bound free amino residues, and the coupling may be carried out in a medium, such as DMF: CH₂Cl₂ (1:1) or in DMF or CH₂Cl₂ alone. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as coupling reagents are N,N′-diisopropyl carbodiimide (DIC), or HBTU combined with HOBt. The success of the coupling reaction at each stage of the synthesis is preferably monitored by the ninhydrin reaction. In cases where incomplete coupling occurs, either the coupling procedure is repeated, or the resin-bound unreacted amino residues are acetylated using Ac₂O/DCM, before removal of the alpha amino protecting group.

Final acylation of the N-terminus of the peptide is done in the same way as the previous couplings, with the difference that the appropriate carboxylic acid is used instead of an amino acid.

4. Removal of the Peptide from the Support Polymer.

When the synthesis is complete, the peptide is cleaved from the support phase. Removal of the peptide from the resin is performed by treatment with a reagent such as liquid hydrogen fluoride which also cleaves all remaining side chain protecting groups.

Suitably, the dried and protected peptide-resin is treated with a mixture consisting of 1.0 mL m-cresol and 10 mL anhydrous hydrogen fluoride per gram of peptide-resin for 60 min at 0° C. to cleave the peptide from the resin as well as to remove all side chain protecting groups. After the removal of the hydrogen fluoride under a stream of nitrogen and vacuum, the free peptides are precipitated with ether, filtered, washed with ether and ethyl acetate, extracted with 50% acetic acid, and lyophilized.

5. Purification

The purification of the crude peptides can be effected using procedures well known in peptide chemistry. For example, purification may be performed on a MacRabbit HPLC system (Rainin Instrument Co. Inc., Woburn, Mass.) with a Knauer UV Photometer and a Kipp and Zonen BD40 Recorder using a Vydac 218TP5010 reversed-phase column (10×250 mm, packed with C18 silica gel, 300 Å pore size, 5 μm particle size) (The Separations Group Inc., Hesperia, Calif.). The column is eluted with a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN in a linear gradient mode (e.g., 30-55% B in 120 min). The eluent is monitored at 220 nm, and fractions are examined by analytical HPLC using a Hewlett-Packard Model HP-1090 liquid chromatograph and pooled to give maximum purity. Analytical HPLC is carried out on a Vydac 218TP52 reversed-phase column (2×250 mm, C18, 300 Å, 5 μm) using isocratic elution with a solvent system consisting of (A) and (B) defined above. The peaks are monitored at 220 and 280 nm. The peptides are judged to be substantially (>95%) pure by analytical HPLC. The expected amino acid composition is also confirmed by amino acid analysis.

D. Pharmaceutical Composition

The peptides of the invention may be administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts. Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, pamoate, malate, ascorbate, tartarate, and the like. Particularly preferred antagonists are salts of low solubility, e.g., pamoate salts and the like. These exhibit long duration of activity.

The compounds of the present invention are suitably administered to subject humans or animals s.c., i.m., or i.v; intranasally or by pulmonary inhalation; or in a depot form (e.g., microcapsules, microgranules, or cylindrical rod like implants) formulated from a biodegradable suitable polymer (such as D,L-lactide-coglycolide), the former two depot modes being preferred. Other equivalent modes of administration are also within the scope of this invention, i.e., continuous drip, depot injections, infusion pump and time release modes such as microcapsules and the like. Administration is in any physiologically acceptable injectable carrier, physiological saline being acceptable, though other carriers known to the art may also be used.

The peptides are preferably administered parenterally, intramuscularly, subcutaneously or intravenously with a pharmaceutically acceptable carrier such as isotonic saline. Alternatively, the peptides may be administered as an intranasal spray with an appropriate carrier or by pulmonary inhalation. One suitable route of administration is a depot form formulated from a biodegradable suitable polymer, e.g., poly-D,L-lactide-coglycolide as microcapsules, microgranules or cylindrical implants containing dispersed antagonistic compounds.

The amount of peptide needed depends on the mode of administration and the intended result. In general, the dosage range is between 1-100 μg/kg of body weight of the host per day.

E. Therapeutic Uses of GH-RH Antagonists

hGH-RH antagonists can be used in treatment of conditions caused by excess growth hormone, for example acromegaly, which is manifested by an abnormal enlargement of the bones of the face and extremities. The GH-RH antagonists may also be used to treat diabetic nephropathy (the main cause of blindness in diabetics) and diabetic retinopathy, in which damage to the eye and kidney respectively is thought to be due to GH.

The hGH-RH antagonists are designed to block the binding and therefore the action of GH-RH, which stimulates the secretion of GH, which in turn stimulates production of IGF-I. GH-RH antagonists may be administered alone or together with somatostatin analogues, a combination which more completely suppresses IGF-I levels. It is advantageous to administer antagonists of GH-RH rather than somatostatin due to the fact that GH-RH antagonists may be utilized in situations where target sites do not have somatostatin receptors.

However, the main applications of GH-RH antagonists are in the field of cancer. This is based on the following considerations: GH-RH antagonists are designed to block the binding and therefore the action of GH-RH, which stimulates the secretion of GH, which in turn stimulates production of insulin-like growth factor I (IGF-I) also called somatomedin-C. The involvement of IGF-I (somatomedin-C) in breast cancer, prostate cancer, colon cancer, bone tumors and other malignancies is well established, and somatostatin analogues alone do not adequately suppress GH and IGF-I levels. A complete suppression of IGF-I levels or secretion is required for a better inhibition of tumor growth. Autocrine production of IGF-I by various tumors could be also under control of GH-RH and might therefore be inhibited by GH-RH antagonists. GH-RH antagonists might also inhibit the production of IGF-I. A more detailed theoretical background of the applications of GH-RH in the field of oncology (cancer) is as follows: The receptors for IGF-I are present in primary human breast cancers, prostate cancers, lung cancers, colon cancers, brain-tumors, pancreatic cancers, and in renal cell carcinomas.

The presence of IGF-I receptors in these tumors appears to be related to malignant transformation and proliferations of these cancers. IGF-I can act as endocrine, paracrine or autocrine growth factor for various human cancers, that is the growth of these neoplasms is dependent on IGF-1. GH-RH antagonists by suppressing GH secretion would lower the production of IGF-I. Since IGF-I stimulates growth of these various neoplasms (cancers), the lowering of circulating IGF-I levels should lead to tumor growth inhibition. It is possible that GH-RH antagonists could also lower paracrine or autocrine production of IGF-I by the tumors, which should also lead to inhibition of cancer proliferation. These views are in accordance with modern concepts of clinical oncology. GH-RH antagonists should be given alone or together with somatostatin analogues and a combination would achieve a more complete suppression of IGF-I levels, elimination of tissue IGF-I levels, e.g., in human osteosarcomas, as well as breast cancer, colon cancer, prostate cancer, and non-small cell lung cancer (non-SCLC).

The advantage of GH-RH antagonists over somatostatin analogues is based on the fact that GH-RH antagonists may be utilized for suppression of tumors which do not have somatostatin receptors, for example human osteogenic sarcomas.

Antagonistic analogs of GH-RH have been shown to suppress growth of various tumors in vivo. This effect is exerted in part through inhibition of the GHRH-GH-IGF-I axis. Nevertheless, autocrine/paracrine control of proliferation by IGF-II is also a major factor in many tumors. Interference with this autocrine growth-stimulating pathway offers an approach to tumor control. Antagonistic analogs of GH-RH, MZ-4-71 {[Ibu⁰, Tyr¹, D-Arg², Abu¹⁵, Nle²⁷]hGH-RH(1-28) Agm} and MZ-5-156 {[PhAc⁰, D-Arg², Abu¹⁵, Nle²⁷]hGH-RH(1-28) Agm} significantly inhibited the rate of proliferation of mammary (MDA-MB-468, ZR-75-1), prostatic (PC-3 and DU-145), and pancreatic (MiaPaCa-2, SW-1990 and Capan-2) cancer cell lines in vitro as shown by colorimetric and [³H]-thymidine incorporation tests, reduced the expression of IGF-II mRNA in the cells and the concentration of IGF-II secreted into the culture medium. The same GH-RH antagonists produced similar results in vivo (inhibition of proliferation and reduction of IGF-II production) for prostate tumors (PC-3, DU-145), renal adenocarcinoma (Caki-I) and non-small cell lung carcinoma (H157). These findings suggest that antagonistic analogs of GH-RH can inhibit tumor growth not only by inhibiting the GHRH-GH-IGF-I axis, but also by reducing the IGF-II production in certain tumor cells, thus interrupting its autocrine regulatory pathway.

The present invention is described in connection with the following examples which are set forth for the purposes of illustration only. In the examples, optically active protected amino acids in the L-configuration are used except where specifically noted.

The following Examples set forth suitable methods of synthesizing the novel GH-RH antagonists by the solid-phase technique.

EXAMPLE I

PhAc⁰-Tyr¹-D-Arg²-Asp³-Ala⁴-Ile⁵-Phe(pCl)⁶-Thr⁷-Asn⁸-Arg⁹-Tyr¹⁰-Arg¹¹-Lys¹²-Val¹³-Leu^{14-Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu22-Gln24-Asp25-Ile26-Nle27-D-Arg28-Har29< /sup>—NH₂ (Peptide 1)}

{[PhAc⁰, D-Arg², Phe(pCl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹]hGH-RH(1-29)NH₂}

The synthesis is conducted in a stepwise manner using manual solid phase peptide synthesis equipment. Briefly, para-methylbenzhydrylamine (MBHA) resin (Bachem, Calif.) (720 mg, 0.50 mmole) is neutralized with 5% DIEA in CH₂Cl₂ and washed according to the protocol described in Table I. The solution of Boc-Har(NO₂)—OH (500 mg, 1.5 mmole) in DMF-CH₂Cl₂ (1:1) is shaken with the neutralized resin and DIC (235 μL, 1.5 mmole) in a manual solid phase peptide synthesis apparatus for 1 hour. After the completion of the coupling reaction is proved by negative ninhydrin test, deprotection with 50% TFA in CH₂Cl₂, and neutralization with 5% DIEA in CH₂Cl₂, the peptide chain is built stepwise by coupling the following protected amino acids in the indicated order on the resin to obtain the desired peptide sequence: Boc-D-Arg(Tos)-OH, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)—OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Lys(2ClZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Lys(2ClZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)-OH, Boc-Arg(Tos)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe(pCl)—OH, Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)—OH, Boc-D-Arg(Tos)-OH, Boc-Tyr(2BrZ)-OH.

These protected amino acid residues (also commonly available from Bachem Co.) are represented above according to a well accepted convention. The suitable protecting group for the side chain functional group of particular amino acids appears in parentheses. The OH groups in the above formulae indicate that each residue's carboxyl terminus is free.

The protected amino acids (1.5 mmole each) are coupled with DIC (235 μL, 1.5 mmole) with the exceptions of Boc-Asn-OH and Boc-Gln-OH which are coupled with their preformed HOBt esters. After removal of the N^α-Boc protecting group from Tyr¹, the peptide is acylated with phenylacetic acid (PhAc) (272 mg, 2 mmole) using DIC (313 μL, 2 mmole).

In order to cleave the peptide from the resin and deprotect it, the dried peptide resin (2.18 g) is stirred with 2 mL m-cresol and 20 mL hydrogen fluoride (HF) at 0° C. for 1 hour. After evaporation of the HF under vacuum, the remnant is washed with dry diethyl ether and ethyl acetate. The cleaved and deprotected peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization, 1.51 g crude product is obtained.

The crude peptide is checked by analytical HPLC using a Hewlett-Packard Model HP-109