WO2010065572A1 - Sstr1-selective analogs - Google Patents

Abstract

Short analogs of SRIF which are selective for SSTR1 in contrast to the other four SRIF receptors comprise a 6-member ring wherein D-Agl(NMe,2Np) and IAmp are flanked by residues having an aromatic side chain end wherein its C-terminus is amidated, inhibit the binding of a universal SRIF radioligand to the cloned human receptor SSTR1, but not to the other receptors. These short selective analogs of SRIF comprise the cyclic hexapeptide: formula (I) where X is H, 4Cl, 4F, 4NO₂, 4NH₂, 4NHCONH₂, 4NHCONHOCH₃, 4NHCONHOCH₂-CH₃ or 4NHCONHOH, where Xaa is Phe, Tyr or iodotyrosine (ITyr) and where the C-terminus is amidated. Radioactive iodinated tyrosine may be one of the two flanking residues, or at the N-terminus, which will also accommodate a conjugating agent or linkage to a cytotoxin.

Description

SSTR1 -SELECTIVE ANALOGS

This application claims priority from U.S. Provisional Application Serial No. 61/119,984, filed December 4, 2008.

This invention was made with Government support under Grant No. 5R01 DK50124 awarded by the National Institutes of Health. The Government has certain rights in this invention.

This invention is directed to peptides related to somatostatin and to methods for pharmaceutical treatment of mammals using such peptides. More specifically, the invention relates to short receptor-selective somatostatin analogs which include particular amino acid substitutions that create receptor-selectivity and increased affinity to the selected receptor, to pharmaceutical compositions containing such peptides, to such peptides complexed with radioactive nuclides or conjugated to cytotoxins, and to methods of diagnostic and therapeutic treatment of mammals using such peptides and their conjugates, particularly peptides that are coupled to chelators and then complexed with radioactive nuclides or otherwise labeled.

BACKGROUND OF THE INVENTION

The cyclic tetradecapeptide somatostatin- 14 (SRIF) was originally isolated from the hypothalamus and characterized as a physiological inhibitor of growth hormone release from the anterior pituitary. SRIF is localized throughout the central nervous system, where it acts as a neurotransmitter and has been shown to both positively and negatively regulate neuronal firing, to affect the release of other neurotransmitters, and to modulate motor activity and cognitive processes.

Somatostatin and many analogs of somatostatin exhibit activity in respect to the inhibition of growth hormone (GH) secretion from cultured, dispersed rat anterior pituitary cells in vitro; they also inhibit GH, insulin and glucagon secretion in vivo in the rat and in other mammals. One suc Ih analog is [D-Trp ; ]-SRIF which has the amino acid i sequence: (cyclo 3-14)H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH, which is disclosed in U.S. Patent No. 4,372,884 (2/8/83). SRIF has also been found to inhibit the secretion of gastrin and secretin by acting directly upon the secretory elements of the stomach and pancreas, respectively, and SRIF is being sold commercially in Europe for the treatment of ulcer patients. SRIF is also known to inhibit the growth of certain tumors.

SRIF induces its biological effects by interacting with a family of membrane-bound structurally similar receptors. Five SRIF receptors have been cloned and are referred to as SSTRl -5. All five receptors bind SRIF and SRIF-28 (an N-terminally extended version of SRIF) with high affinity; cell lines bearing these cloned receptors are available to test SRIF analogs for binding affinity, selectivity and functional effects. Studies have now shown that different receptor subtypes mediate distinct functions of SRIF in the body.

A cyclic SRIF analog, variously termed SMS-201-995 and Octreotide, i.e. H-D-Phe- c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol is being used clinically to inhibit certain tumor growth. Analogs complexed with ¹¹¹In or the like are also used as diagnostic agents to detect SRIF receptors expressed in cancers.

Octreotide, angiopeptin and other clinically used SRIF analogs interact significantly with three of the receptor subtypes, i.e. SSTR2, SSTR3 and SSTR5. A comprehensive review of SRIF and its receptors is found in Patel, Y.C. "Somatostatin and its receptor family", Front. Neuroendocrinal, 1999, 20, 157-198. U.S. Patent No. 7,238,775 (July 3, 2007) discloses SSTR4-selective analogs of SRIF, and Patent No. 6,579,967 discloses SSTR3-selective synthetic analogs. U.S. Patent No. 5,750,499 (May 12, 1998) and Patent No. 7,019,109 disclose SRIF analogs which are selective for SSTRl. It has been reported that in medullary thyroid carcinoma, calcitonin secretion and gene expression can be reduced by treatment with SSTRl -selective agonists and that such may be able to inhibit endothelial activities, suggesting a potential therapeutic utility for administration of SSTRl -selective agonists in the proliferative diseases involving angiogenesis. Although numerous reports on the localization, physiological and therapeutic functions of SSTRl have been published, it is still not clear which is its main function and the main related pathology resulting from over- or under-expression. The design of more potent and more highly selective peptide agonists for SSTRl that are radio-labelable and which have greater metabolic stability in biological fluids than the native hormone should help to further understand SSTRl -related biology and pathobiology. Such shortened peptide agonists are advantageous for production and other considerations, and such can be very important economically.

SUMMARY OF THE INVENTION

Certain short peptide analogs of SRIF have now been discovered which are highly selective for SSTRl in contrast to the other cloned human SRIF receptors, i.e. SSTR2-5 and which have high binding strength. Building upon the inclusion of an alkylated aminomethyl Phe within the ring of a SRIF analog that binds to SSTRl, it has now been found that high selectivity and high binding strength can be obtained in much shorter analogs by amidating the C-terminus and by incorporating an N^methylated and acylated residue adjacent the aminomethyl Phe residue. As a result, short peptides have now been created that bind strongly and selectively to cloned SSTRl and exhibit agonist properties. One such peptide can be radioiodinated or otherwise radiolabeled while retaining its desirable biological properties. These novel peptide agonists are useful in determining the tissue and cellular expression of the receptor SSTRl and its biological role in the endocrine, exocrine and nervous system, as well as in regulating certain pharmacological functions without the accompanying side effects which have heretofore been characteristic of administering the native releasing factor SRIF or an analog which binds significantly to two or more of SSTRl -5. These short SRIF analog peptides, when radiolabeled, can be used in scintigraphy in order to locate, i.e. localize, tumors expressing these receptors, either in vitro or in vivo; other labeling as well known in this art, e.g. fluorescent, can alternatively be used. With an appropriate chelated radioligand, these analogs can be turned into radiopharmaceuticals which are suitable for radionuclide therapy in treatment of such tumors; alternatively, they can be covalently joined to a cytotoxic moiety using an appropriate covalent conjugating agent, e.g. glutaraldehyde or one which binds via a disulfide linkage.

These short peptide agonists inhibit the binding of ¹²⁵I-[Tyrⁿ]SRIF and ¹²⁵I- [Leu⁸,D-Trp²²,Tyr²⁵]SRIF-28 to the cloned human receptor SSTRl, but they do not bind with high affinity to SSTR2, SSTR3, SSTR4 or SSTR5. As such, these SSTRl specific analogs may be used to treat SSTRl -mediated physiopathologies; there is evidence that intimal hyperplasia (IH) may be treated in this way. These analogs to which ⁹⁹Tc, ¹¹¹In or ⁹⁰Y, for example, has been chelated by a linker, such as DOTA or DTPA, or to which other complexing or conjugating agents are linked to the N-terminus for the purpose of attaching moieties, e.g. cytotoxins, are useful for diagnostic or therapeutic purposes. Conjugating agent is used herein to broadly refer to this class of well known chelating, complexing or otherwise covalently bound agents that serve to link desired moieties to peptides.

These short SRIF agonists bind selectively and with high affinity to SSTRl; by selectively binding is meant that they exhibit an IC₅₀ (or K_D) with SSTRl that is about 10% or less of the IC₅₀ which they exhibit with respect to each of the other four SRIF receptors; by high affinity is meant an IC₅₀ of not greater than about 10 nanomolar. Of course, the greater the differentials, the more selective the analog is. These short SRIF analogs can also be readily labeled and effectively used in radionuclide and cytotoxic therapy; for example, they are useful in localizing such receptor in the body and in diagnosing the locations of tumors, particularly prostate cancers, sarcomas and neuroendocrine tumors. As radionuclide therapeutic agents, they are considered to be particularly useful in destroying tumors expressing SSTRl receptors.

In one particular aspect, the invention provides novel cyclic short somatostatin (SRIF) analog peptide agonists which selectively bind the SRIF receptor SSTRl, said peptide comprising the cyclic amino acid sequence:

Cys-Phe(X)-D-Agl(NMe, 2Np)-IAmp-Xaa-Cys wherein Xaa is Phe, Tyr or iodotyrosine (ITyr) and the C-terminus is amidated, where X is H, 4Cl, 4F, 4NO_{2 ,} 4NH₂, 4NH-CONH₂, 4NHCONHOCH₃, 4NHCONHOCH₂-CH₃ or 4NHC0NH0H; D-AgI(NMe, 2Np) stands for N^Me, 2-naphthoyl aminoglycine and IAmp stands for 4-(N-isopropyl)- aminomethy lphenylalanine .

In another particular aspect, the invention provides pharmaceutical compositions comprising the novel peptides and a pharmaceutically acceptable carrier which can be administered in an amount effective to treat IH or another SSTRl -mediated physiopathology by reaching tissue having SSTRl receptors and activating said receptors, or administered in an effective amounts so as to selectively bind to cells having SSTRl cells and thereby provide a detectable signal at the location thereof, or administered in an amount which is effective to destroy cells containing SSTRl via a radioactive nuclide or a cytotoxin coupled to the novel peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The nomenclature used to define the peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the right. The standard 3 -letter abbreviations to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated, e.g. Ser = L-serine. The Cys residue at or adjacent the N-terminus may be in either the L- or D-isomer form without affecting binding affinity or function. By "cyclo" or "c" is meant that a cyclizing bond is present between the side chains of the two cysteine residues.

SRIF analog peptides are herein provided that have both a selective affinity for the SRIF receptor SSTRl and a high affinity for SSTRl, i.e. an IC_5O that is preferably less than 10 nM. These short SRIF peptide analogs have a ring of six amino acid residues formed by a disulfide linkage between Cys residues at the 1 and 6-positions wherein the central two residues, i.e. in the 3 and 4-positions, respectively comprise a D-isomer aminoglycine having a substituted β-amino group in the 3 -position, and an L-isomer alkylated aminomethylphenylalanine (Amp), preferably (isopropyl)Amp, in the adjacent 4-position. These two residues are flanked by a pair of amino acid (AA) residues having aromatic side chains, preferably either phenylalanine or tyrosine, substituted or unsubstituted. More preferably, Tyr or ITyr is present in the 5-position, and the C-terminus is amidated. By amidated is meant that the residue at the C-terminus carries an amide group, symbolized by -NH₂, or a substituted amide group symbolized by -NHR where R is lower alky, preferably, methyl, ethyl or propyl. It has long been appreciated that, from an economic standpoint, the ability to provide a shorter peptide that exhibits the same or substantially the same pharmacological properties as a longer peptide is of significant value where such peptides must be produced by chemical synthesis, either solid-phase synthesis or solution-phase synthesis. For this reason, steps have traditionally been taken to see whether the effects of isolated peptide hormones can be essentially duplicated by shortening the N-terminus or the C-terminus of the peptide. With respect to somatostatin, other than elimination of the 2-residue tail at the N-terminus, the path was unclear as to the effect of shortening, and it was particularly unclear what characteristics in a peptide chain would provide short SRIF peptide analogs that would be fully selective for a single receptor, and more particularly for SSTRl . It was known that the octapeptide Otreotide, having a 6-residue ring, mimicked effects of SRIF in a certain number of aspects; it was known that this 6-member ring compound interacted significantly with SSTR2, SSTR3 and SSTR5 but showed little affinity for SSTRl. Moreover, when the C-terminus of Otreotide was amidated, there was no improvement in affinity of the octapeptide for SSTRl.

Despite these unfavorable initial results, efforts persisted to try to produce a potent, short SRIF analog that would very selectively bind to SSTRl . It has now been surprisingly found that a cyclic ring of only six amino acid residues will exhibit these desired characteristics if the four residues situated between the cysteine residues (which form the ring by creation of a disulfide bond between their side chains) comprise two particular synthetic AA residues flanked by amino acids having aromatic side chains, and if the C- terminus of the cyclic hexapeptide is amidated. As mentioned above, the residue in the 3- position is a D-isomer of a substituted aminoglycine, more particularly one where the β- amino group is both methylated and acylated, preferably with 2-naphthalene carboxylic acid. The adjacent residue in the 4-position is an aminomethylphenylalanine (Amp) which is C₂-C₅ alkylated, preferably with isopropyl, i.e. 4-(N-isopropyl)-aminomethyl Phe. The C-terminus may be either amide or substituted amide, e.g. ethylamide. Optionally, a threonine or a naphthylalanine residue may be added at the C-terminus, e.g. Thr-NH₂ or 2NaI-NH₂, to somewhat improve the potency of the analog as a result of its binding with a stronger affinity to SSTRl; however, such may cause some lessening of the differential between its affinity for SSTRl and for SSTR4. Tyr may be slightly preferred over Phe for the 5 -position residue, and radioiodinated Tyr (ITyr) acts as a tracer without significantly lessening its selectivity for SSTRl or its binding affinity thereto. Optionally, L- or D-Tyr can be added at the N-terminus, which may also be radioiodinated; such addition may also improve potency of the SRIF agonist but may also exhibit a somewhat increased affinity for SSTR4.

As indicated above, a conjugating/complexing agent can be linked to the α-amino group of Cys at the N-terminus of the peptide analog, which agent is capable of joining thereto a radioactive nuclide or a cytotoxin. These conjugating/complexing agents may be any of those presently used in this art which covalently bond to an α-amino group. They may be designed to link, as by chelation, to a radioactive metal or to covalently bind to a cytotoxin, such as saporin, gelonin, ricin A chain, etc.

The effective SRlF cyclic analog agonists comprise the amino acid sequence: Cys-Phe-D- AgI(NMe, 2Np)-IAmp-Xaa-Cys wherein Xaa is Phe, Tyr or iodotyrosine (ITyr) and the C-terminus is amidated, where X is H, 4Cl, 4F, 4NO₂ , 4NH₂, 4NH-CONH₂, 4NHCONHOCH₃, 4NHCONHOCH₂-CH₃ or 4NHC0NH0H, where D-AgI(NMe, 2Np) stands for D-N^Me, 2-naphthoyl aminoglycine and where IAmp stands for 4-(N-isopropyl)- aminomethylpheylalanine.

By Amp is meant (aminomethyl)phenylalanine where the methyl group with its amino substitution should be understood to be in the 4- or para-position on the phenyl ring. By IAmp is meant (N-isopropyl-aminomethyl)phenylalanine, where the 4-aminomethyl group is alkylated with an isopropyl group; whereas in EAmp, the alkylation is with an ethyl group. As used herein, the term "lower alkyl" refers to a straight or branched chain, saturated hydrocarbon group having from 1 to 6 carbon atoms such as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, isopentyl, n-pentyl and n- hexyl. By Me is meant methyl. By Np is meant naphthoyl. As used herein, naphthoyl is inclusive of 1- and 2-naphthoyl, with 2-naphthoyl being preferred. By NaI is meant 3-(2- naphthyl)-alanine. By SRIF is meant the 14-residue cyclic peptide, somatostatin. When Tyr appears in the 5-position, it may be radioiodinated or otherwise labeled; by ITyr is meant radioiodinated tyrosine.

The C-terminus is amidated as described hereinbefore. The N-terminus may be modified in various ways without significantly adversely effecting the binding affinity, all of which modifications in these cyclic peptides are considered to be included as a part of the peptides of the overall invention. For example, L- or D-Tyr or Phe, DPhe, Phe(X), DPhe(X) (X = Cl, F, NO₂) can be added. A variety of additions may also be made to the N- terminal amino acid in the form of conjugating agents which can be then used to link a desired moiety to the peptide. For example, chelating agents, such as DTPA, DOTA, HYNIC and P₂S₂-COOH may be attached; alternatively, a cytotoxin may be covalently linked thereto via a suitable linker well known in this art if desired.

Although SSTRl was the first somatostatin receptor cloned, identification of its biological and pharmacological properties has lagged somewhat behind the other SRIF receptors because of the lack of ligands which are significantly selective for SSTRl. Relevant peptides to date are disclosed in the '499 and '109 patents and in the following literature articles: (1) George Liapakis et al, "Development of a Selective Agonist at the Somatostatin Receptor Sutype SSTRl", The Journal of Pharmacology and Experiemental Therapeutics, 1996, VoI 276, No. 3, Pages 1089-1094. (2) Jean Claude Reubi et al, "A selective analog for the somatostatin sstl -receptor subtype expressed by human tumors", European Journal of Pharmacology, 1998, Vol. 345, Pages 103-110. (3) Longchuan Chen et al, "Structural Basis for the Binding Specificity of a S STRl -Selective Analog of Somatostatin", Biochemical and Biophysical Research Communications, 1999, Vol. 258, Pages 689-694. (4) Jean Rivier et al, "Potent somatostatin undecapeptide agonists selective for somatostatin receptor 1 (sstl)", Journal of Medicinal Chemistry, 2001, VoI 44, Pages 2238-2246. (5) Judit Erchegyi et al, "Somatostatin Receptor 1 Selective Analogues: 2. N^α- Methylated Scan", Journal of Medicinal Chemistry, 2005, Vol. 48, Pages 507-514. The peptides of the invention are believed to be improvements over those disclosed in the '109 patent and in these articles, and they are expected to be very helpful in determining the many functional roles of this receptor and in selectively binding only this SRIF receptor and not the others. They are also expected to be valuable in treating IH, and they are expected to be particularly valuable in SRIF receptor-targeted scintigraphy and radionuclide therapy. For a number of reasons it is considered advantageous to administer peptide ligands of this character, rather than nonpeptide ligands.

Selectivity for binding of the analog peptides of the invention to SSTRl is demonstrated by testing their interaction with the five different cloned human SRIF receptors as described in detail hereinafter. Generally, recombinant cells expressing the receptor are washed and homogenized to prepare a crude protein homogenate that is frozen, embedded and then cut in 10-20 μm thin sections, as known in this art. Candidate substances, i.e. potential SRIF agonists or antagonists, are incubated with the tissue sections, and the interaction between the candidate substance and the receptor polypeptide is monitored. The short SRIF analog peptides of the invention bind substantially only to SSTRl , and their binding exhibits high affinity. Receptor binding assays are performed on cloned SRIF receptors as generally set forth in Reubi, J.C. et al., J. Clin. Endocrinol. Metab., 63, 433-438 (1986) and in Reubi, J. C. et al., Eur. J. Nucl. Med. 2000, 27, 273-282. Using such assays, one can generate K_D values which are indicative of the concentration of a ligand necessary to occupy one-half (50%) of the binding sites on a selected amount of a receptor or the like, or alternatively, competitive assays can generate IC_5O values which are indicative of the concentration of a competitive ligand necessary to displace a saturation concentration of a target ligand being measured from 50% of binding sites. The cyclic hexapeptide

I I

H-c[Cys-Phe-DAgl(NMe,2Np)-IAmp-Tyr-Cys]-NH₂ inhibits the binding to SSTRl of an iodinated SRIF-28 ligand that is known to have a strong affinity for all five receptors. Testing shows that it binds to the cloned human SSTRl with an IC₅₀ of about 6 nM, while this short SRIF analog peptide does not bind strongly to either human SSTR3 and SSTR4 and does not bind to SSTR2 or SSTR5 at a concentration below 1,000 nM. The tyrosine residue in position-5 may be radioiodinated without significantly changing the selective affinity of the ¹²⁵I-Tyr analog so it serves as an excellent tracer for SSTRl. The similar

I 1 analog H-c[Cys-Phe-DAgl(NMe,2Np)-IAmp-Phe-Cys]-Thr-NH₂ inhibits binding of iodinated SRIF-28 to SSTRl while itself binding even more strongly with an IC₅₀ of about 1.0 nM, while still not binding to receptors SSTR2 or SSTR5 at concentrations below 1,000 nM; however, it does show slightly higher affinity for SSTR4. These SRIF analogs that selectively show high affinity to SSTRl are considered to be particularly useful in the treatment of SSTRl -mediated physiopathologies and in combating tumors by carrying radionuclides or cytotoxins to the sites of these receptors but not to other SRIF receptors.

As hereinbefore indicated, SSTRl mRNA has been detected in a variety of tumors. However, it is presently not known whether SSTRl plays a major role in tumor growth regulation and, if it does, whether it mediates stimulation or inhibition. Therefore, at this time, it is difficult to foretell whether a selective SSTRl antagonist would have a beneficial role for long-term treatment of tumors. However, the use of SRIF analogs selective for SSTRl that bind strongly thereto, and that are long-acting can be effectively used to kill such tumors via radionuclide or cytotoxic therapy. To date the use of Octreotide in the treatment of such tumors has not been considered to be satisfactorily effective particularly because of its selectivity to 3 of the 5 receptors, i.e. SSTR2, 3 and 5.

SSTRl has been reported to couple to a tyrosine phosphatase, and stimulation of this enzyme is believed to mediate anti-proliferative effects of SRIF via activation of this receptor. SSTRl niRNA has been detected in a number of tumors. The ability of SSTRl to mediate anti-proliferative effects of SRIF renders SSTRl -selective SRIF agonists effective as therapeutic treatment agents for treating those cancers, such as prostrate cancers and sarcomas wherein the malignant tissues express this receptor.

A particularly important advantage of SSTRl -selective agonists as anti-cancer agents may be their continued effectiveness after prolonged use. For example, continuous use of SMS-201-995 in the treatment of tumors is considered to be hindered by rapid desensitization of SSTR2, SSTR3 and SSTR5, the receptors with which this peptide can interact; in fact, all 3 of these receptors have been reported to rapidly desensitize. In contrast, studies suggest that SSTRl may be more resistant to agonist-induced regulation than the other receptors. As a result, the SSTRl -selective peptide agonists of the invention are considered to have prolonged anti-proliferative actions and should therefore exhibit improved effectiveness in treating SSTRl -mediated cancers, compared to the commercially available SRIF analogs presently used as anti-cancer agents that have low affinity for SSTRl.

The SRIF analogs of the present invention are considered to be useful in combating cancers which express SSTRl and in combating SSTRl -mediated physiopathologies. They are also considered to be most useful in scintigraphy to determine the distribution of cells and tissues expressing this receptor in the brain and in the endocrine and exocrine systems, and also in identifying selective functions of this receptor in the body.

Labeled SRIF analogs of the invention are also considered to be useful in drug- screening assays to screen for new effective peptide and nonpeptide agents which will bind with high affinity to SSTRl and which may be either highly effective agonists or antagonists for treating GI track motility. Once a known ligand for the receptor SSTRl is in hand, one can obtain a baseline activity for the recombinantly produced receptor. Then, to test for inhibitors or modifiers, i.e. antagonists of the receptor function, one can incorporate a candidate substance into a test mixture to test its effect on the receptor. By comparing reactions which are carried out in the presence or absence of the candidate substance, one can then obtain information regarding the effect of the candidate substance on the normal function of the receptor. The cyclic SRIF analogs described in the following examples are agonists which can be also employed to selectively stimulate the inhibitory activity of somatostatin at SSTRl .

The peptides of the present invention can be synthesized by classical solution-phase synthesis, but they are preferably synthesized by solid-phase technique, as described in United States Patent No. 5,750,499 issued May 12, 1998, the disclosure of which is incorporated herein by reference.

The following Examples illustrate the syntheses of several short SRIF analog cyclic peptides embodying various features of the invention, together with the syntheses of protected amino acids for use in such peptide syntheses. All of these peptides include a D- isomer amino acid residue. In each peptide, "cyclo" should be understood to indicate that the two cysteine residues in the peptide chain are joined by a cyclizing disulfide bond. Example 1

Synthesis ofN^αBoc-(4-isopropylaminomethyl)phenylalanine The synthesis of L-N^αBoc-N⁴-Cbz-(4-isopropylaminomethyl)phenylalanine, which is referred to by the shorthand nomenclature as Boc-IAmp(Z), is carried out as follows: N^αBoc-(4-aminomethyl)phenylalanine (Boc-Amp) (15.3 g, 52 mmol) is dissolved in acetone (200 mL), and molecular sieves (6.0 g, 4A) are added to the solution in a 500 mL Parr hydrogenation vessel. The mixture is purged with N₂ for 10 minutes; then Pd/C 10% (600 mg) is added and hydrogenated. A reductive alkylation reaction occurs and is monitored by HPLC; it is carried out for about 26 hours. After filtration to remove the catalyst and molecular sieves and evaporation of the solvent, the desired intermediate N^αBoc-(4-isopropylaminomethyl)phenylalanine is obtained as a viscous liquid.

The product is then Cbz-protected using benzyl chloroformate (Z-Cl, 8.6 mL, 60 mmol) in a mixture of THFZH₂O(1 : 1,200 mL) at pH = 9.5. A good yield of N^αBoc-N⁴-Cbz- (4-isopropylaminomethyl)phenylalanine is obtained: 17.5 g (37 mmol, 71.4%); m.p. 39- 42⁰C; α_D = +5.2° (c = 1, MeOH, t = 2O⁰C).

Example 2

Synthesis of

The synthesis of unresolved N^Boc-N^Me^-naphthoyl aminoglycine, which is referred to by the shorthand nomenclature as Boc-N^(BMeAgl(2Np) or Boc-Agl(NMe,2Np)- OH, may be carried out either prior to the peptide synthesis or while the peptide intermediate is on the resin. Boc-D/L-Agl(Fmoc)-OH, prepared by the synthesis set forth in Qasmi, et al., Tetrahed, Lett, 34:3861-3862 (1993). Then unresolved N^αFmoc-D/L-

is synthesized using the procedure set forth in Jiang, et al., Orthogonally protected N^βmethyl-substituted-aminoglycine, Protein and Peptide Lett., 3:219-224 (1996). The Fmoc protection can be removed from the monomer (or when a part of the peptide resin) with 20% piperidine in NMP, or the Boc protection can be removed from the monomer (or when a part of the peptide resin) with TFA in DCM and naphthoyl is then introduced by reaction of the free side chain secondary amino group with naphthoyl chloride.

Synthesis of Somatostatin Analogs

Example 3

A somatostatin agonist having the structure:

(cyclo)H-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂ is synthesized by the solid phase methodology in a stepwise manner on a 4-methylbenzhydrylamine (MBHA) resin generally as described in Example 1 of U.S. Patent No. 5,807,983. Such solid-phase approach with Boc chemistry was carried out either manually or on a CS-Bio Peptide Synthesizer Model CS536. 4-Methylbenzhydrylamine (MBHA) resin with a capacity of 0.4 mequiv/g was used. Couplings of the protected amino acids were mediated by diisopropylcarbodiimide (DIC) and (HOBt) in DMF for 1 h and monitored by the qualitative ninhydrin test. A 3 -equivalent excess of the protected amino acids based on the original substitution of the resin was generally used. For the 4-position residue, the monomer synthesized in Example 1 , i.e. N^α(Boc)-4-isopropylaminomethyl Phe(Z) is coupled into the chain. For the 3 -position residue, the monomer synthesized in Example 2 is so coupled. Boc removal was achieved with trifluoroacetic acid (60% in CH₂Cl₂, 1-2% ethanedithiol or m-cresol) for 20 min. An isopropyl alcohol (1% m-cresol) wash followed TFA treatment and then successive washes with triethylamine solution (10% in CH₂Cl₂), methanol, triethylamine solution, methanol and CH₂Cl₂ completed the neutralization sequence. After Fmoc-D/LAgl(N^βMe,Boc)-OH was coupled, D/L-Agl(N^βMe,2naphthoyl) residue was formed on the resin. The Boc protecting group was removed with TFA; 3- equivalent 2-naphthoyl chloride and 3 -equivalent DIPEA were used to acylate the free secondary amino group of the side chain. Removal of the N^α-Fmoc protecting group with 20% piperidine in DMF in two successive 5 and 15 min treatments was followed by the standard elongation protocol until the completion of the peptide.

The peptides were cleaved from the resin support with simultaneous side chain deprotection by anhydrous HF containing the scavengers anisole (10% v/v) and methyl sulfide (5% v/v) for 60 min at 0 ⁰C. The diethyl ether precipitated crude peptides were cyclized in 75% acetic acid (200 mL) by addition of iodine (10% solution in methanol) until the appearance of a stable orange color. Forty minutes later, ascorbic acid was added to quench the excess iodine.

Purification and characterization of the analogs. The crude, lyophilized peptides were purified by preparative RP-HPLC on a 5 cm x 30 cm cartridge, packed in the laboratory with reversed-phase Vydac C₁₈ silica (15-20 μM particle size, 300 A) using a Waters Prep LC 4000 preparative chromatograph system. The peptides eluted with a flow rate of 100 mL/min using a linear gradient of 1% B per 3 min increase from the baseline % B. Eluent A = 0.25 N TEAP pH 2.25 in water, eluent B = 60% CH₃CN, 40% A. All peptides were subjected to a second purification step carried out with eluents A = 0.1% TFA in water and B = 60% CH₃CN/40% A on the same cartridge using a linear gradient of 1% B per min increase from the baseline %B. The collected fractions were screened by analytical RP-HPLC, and the fractions containing the product were pooled and subjected to lyophilization. The purity of the final peptide was determined by analytical RP-HPLC performed with a linear gradient using 0.1 M TEAP pH 2.5 in water as eluent A and 60% CH₃CN/40% A as eluent B on a Hewlett-Packard Series II 1090 Liquid Chromatograph connected to a Vydac Qg column (0.21 x 15 cm, 5 μm particle size, 300 A pore size). The products were then analyzed by capillary zone electrophoresis (CZE), and mass spectra (MALDI-TOF-MS) were measured on an ABI-Perseptive DE-STR instrument. The observed monoisotopic (M + H)⁺ values of each peptide corresponded with the calculated (M + H)⁺ values. Because the L and D enantiomers of Fmoc-D/LAgl(NMe,Boc) used for the synthesis of peptides were not resolved initially, two diastereomers were generated, isolated, characterized, and tested. Separation of the L- from the D-Agl-containing peptides was achieved using RP-HPLC.

Determination of the stereochemistry of AgI in the peptides.The absolute configuration of the AgI was deduced from enzymatic hydrolysis of each of the two enantiomers with Aminopeptidase M. Peptides having an L-amino acid at the N-terminus and IAmp within the ring were digested with Aminopeptidase M, which is a metalloprotease, and can hydrolyze peptides at a free α-amino group of L-amino acids to determine the absolute configuration of AgI. The treatment of one enantiomer with Aminopeptidase M at room temperature for 48 hours resulted in many very hydrophilic products followed by RP-HPLC, indicating that the peptides had been completely hydrolyzed, which is evidence that this analog contained the L-enantiomer of AgI in its sequence. The other enantiomer was hydrolyzed into only two products indicating that these peptides contained the D-enantiomer of AgI in its sequence, resulting in more resistance to the enzymatic hydrolysis.

I I

The desired cyclic peptide (cyclo)H-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys- NH₂ is obtained which appears to be greater than 87% pure on capillary zone electrophoresis. MS analysis shows an [M+H]⁺ monoisotropic mass of 990.18 Da, which compares favorably to the calculated mass of 989.40 Da. The peptide is hereinafter referred to as Peptide No. 3.

Examples 3A

A portion of the product Peptide 3 above is taken and subjected to iodination, as well known in this art, to iodinate the Tyr residue in the 5-position and create a tracer. The iodinated compound is thereafter referred to as Peptide 3A.

Example 4

The synthesis described in Example 3 is repeated with one change. Boc-Thr(Bzl) is coupled to the resin prior to the first Cys residue. Elongation of the chain is then carried out as in Example 3, and cleavage, deprotection, cyclization, purification, separation and assignment the stereochemistry of the L and D-AgI containing analogs are also carried out as in Example 3. A purified cyclic peptide having the formula:

I I

(cyclo)H-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-Thr-NH2 results and is referred to as Peptide No. 4.

MS analysis of Peptide No. 4 shows an [M+H]⁺ mass of 1091.44 Da, which compares favorably with the calculated mass of 1090.45 Da. It is selective for and has high binding affinity to SSTRl . Example 5

The synthesis described in Example 4 is repeated with one change. N^αBoc-Phe is used instead of N^αBoc-Tyr(2BrZ) in position-5. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization, and purification are carried out as in Example 3. The purified cyclic peptide has the formula:

I I

(cyclo)H-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Phe-Cys-Thr-NH₂ and is referred to as Peptide No. 5.

MS analysis of Peptide No. 5 shows an [M+H]⁺ mass of 1075.60 Da, which compares favorably with the calculated mass of 1074.45 Da. It is selective for and has high binding affinity to SSTRl.

Example 6

The synthesis described in Example 5 is repeated with one change. Elongation of the chain by one residue is carried out by coupling N^α Boc-D-Tyr(2BrZ) at the N-terminus. Cleavage, deprotection, cyclization and purification are carried out as in Example 3. The purified, cyclic peptide has the formula:

(cyclo)H-D-Tyr-Cys-Phe(X)-D-Agl(NMe,2Np>-IAmp-Phe-Cys-Thr-NH₂ and is referred to as Peptide No. 6. MS analysis of Peptide No. 6 shows an [M+H]⁺ mass of 1238.52, which compares favorably with the calculated value of 1237.52.

This peptide is readily radioiodinated with ¹²⁵I to provide ligands for use in competitive drug screening assays. Following radioiodination, Peptide No. 6 continues to bind strongly and selectively to SSTRl . Example 7

The synthesis described in Example 3 is repeated with one change. DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid), a polyaminopolycarboxylic chelator, is added to the N-terminus of the peptide.

This conjugation step is carried out (a) as the last step of the solid-phase peptide synthesis, or it is carried out (b) in solution phase after synthesis, cleavage, cyclization and purification of the peptide. The reagent l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide) ester.3CH₃COOH.HPFg used in both procedures is purchased from Macrocyclics Inc. (Dallas, TX, USA).

(a) After deprotecting the N-terminal α-amino group of the peptide with trifluoroacetic acid (TFA), a 10-fold excess of DOTA-NHS -ester in dimethylformamide (DMF) in the presence of diisopropylethyl amine (DIPEA) is added to the resin and stirred at room temperature until the reaction is over, as checked by qualitative ninhydrin test. After the reaction is complete, the peptide is cleaved from the resin by HF, cyclized and purified as in Example 3.

(b) After cleavage of the peptide from the resin and deprotection of the side chain protecting groups by HF, followed by cyclization and a RP-HPLC purification, the addition of DOTA to the N-terminus of the peptide is carried out in solution. To a solution of (cyclo)Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂ (25 mg, -25 μM), in dry N₃N- dimethylformamide (DMF, 1800 μl), is added a solution of DOTA-NHS-ester (40 mg, -50 μM) in DMF (160 μl) and N,N'-Diisopropylethylamine (DIPEA) (43 μl, 25 μM). The mixture is stirred at room temperature for several hours. The progress of the reaction is followed by analytical HPLC. After completion of the reaction, a preparative RP-HPLC purification is performed affording the pure peptide with the formula (cyclo)(DOTA)Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂. The mass of the final DOTA-peptide-conjugate is 1375.58. The peptide is referred to as Peptide No. 7. The peptide binds strongly and selectively to SSTRl .

Example 8

The synthesis described in Example 7 is repeated with one change. A different polyaminopolycarboxylic chelator moiety, namely DTPA (Diethylenetriamine pentaacetic acid), is added to the N-terminus of the peptide.

This conjugation step is carried out (a) as the last step of the solid-phase peptide synthesis, or it is carried out (b) in solution phase after synthesis, cleavage, cyclization and purification of the peptide. The reagent DPTA-tetra-tert-butyl ester (Diethyl enetriamine- N,N,N",N"-tetra-tert-butyl acetate-N'-acetic acid) used in both procedures is purchased from Macrocyclics Inc. (Dallas, TX, USA).

(a) The DTPA-tetraester is coupled to the N-terminus of the resin-bound peptide using a 10 equivalents of DTPA-tetraester, 10 equivalents of TBTU and 10 equivalents of DIPEA in DMF. The reaction mixture is stirred until the reaction is over, as checked by qualitative ninhydrin test. After the reaction is complete, the peptide is cleaved from the resin by HF, cyclized and purified as in Example 3.

(b) After cleavage of the peptide from the resin and deprotection of the side chain protecting groups by HF, followed by cyclization and a preparative RP-HPLC purification, the addition of DPTA to the N-terminus of the peptide is carried out in solution. To a solution of (cyclo)Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂ (-25 μM), in dry N₅N- dimethylformamide (DMF), is added a solution of -50 μM of DTPA-tetraester, -50 μM of TBTU and 100 μM of DIPEA in DMF. The mixture is stirred at room temperature for a several hours. The progress of the reaction is followed by analytical HPLC. After completion of the reaction and removal of the tert-butyl protecting groups of DTPA with TFA, a preparative RP-HPLC purification is performed affording the pure peptide with the formula (cyclo)(DPTA)Cys-Phe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂. The mass of the final DPTA-peptide-conjugate is 1364.53. The peptide is referred to as Peptide No. 8. The peptide binds strongly and selectively to SSTRl .

Example 9

The synthesis described in Example 5 is repeated with two changes. N^αBoc-2Nal is used instead of N^αBoc-Thr(Bzl) as the first residue coupled to the resin, and D-Cys is used instead of L-Cys for the residue at the N-terminus. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization, and purification are carried out as in Example 3. The purified cyclic peptide has the formula:

(cyclo)H-D-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Phe-Cys-2Nal-NH₂ and is referred to as Peptide No. 9.

MS analysis of Peptide No. 9 shows [M+H]⁺ mass of 1238.52 Da, which compares favorably with the calculated mass of 1237.52 Da. It is selective for and has high binding affinity to SSTRl .

Example 10

The synthesis described in Example 9 is repeated with one change. N^αBoc- Tyr(2BrZ) is coupled to the D-Cys residue at the N-terminus. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization, and purification are carried out as in Example 3. The purified cyclic peptide has the formula: (cyclo)H-Tyr-D-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Phe-Cys-2Nal-NH₂ and is referred to as Peptide No. 10.

MS analysis of Peptide No. 10 shows an [M+H]⁺ mass of 1334.81 Da, which compares favorably with the calculated mass of 1333.55 Da. It is selective for and has good binding affinity to SSTRl .

Example 11

The synthesis described in Example 3 is repeated with one change. N^αBoc-4ClPhe is used instead of N^αBocPhe in the 2-position. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization and purification are carried out as in Example 3. A purified cyclic peptide having the formula:

I I

(cyclo)H-Cys-4ClPhe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂ results and is referred to as Peptide No. 11. The mass of Peptide No. 11 is 1023.35. Peptide No. 11 binds strongly and selectively to SSTRl.

Example 12

The synthesis described in Example 3 is repeated with one change. N^αBoc-4FPhe is used instead of N^αBoc-Phe in the 2-position. Following removal of the Boc group at the N- terminus, HF cleavage, deprotection, cyclization and purification are carried out as in Example 3. A purified cyclic peptide having the formula:

(cyclo)H-Cys-4FPhe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH₂ results and is referred to as Peptide No. 12. The mass of Peptide No. 12 is 1007.38. Peptide No. 12 binds strongly and selectively to SSTRl. Biological testing

To determine their SSTR-binding properties, the peptides were tested for their ability to bind to cryostat sections of a membrane pellet of cells expressing the five human SRIF receptor subtypes. For each of the tested compounds, complete displacement experiments were carried out with the universal SRIF radioligand [Leu⁸,DTφ²²,¹²⁵ITyr²⁵]SRIF-28 (¹²⁵I-[LTT]SRIF-28). The results are shown in Table 1. The short SRIF analogs were evaluated for their agonist/antagonist properties using a reporter gene assay that determines the biological activity of the human SSTRl in CCL39- SSTRl -Luci cells constitutively expressing the human SSTRl as well as the luciferase gene under the control of the serum response element (SRE). The SRE is regulated by transcription factors and is activated by many extracellular signals including ligands acting at G-protein-coupled receptors. It has been shown that upon ligand binding SSTRs mediate the increase of luciferase expression via SRE in a reporter gene assay. Stimulating CCL39- SSTRl -Luci cells with somatostatin analogues activates the luciferase gene in a dose dependant manner.

The potencies of certain SRIF analogs to inhibit radioligand binding of ¹²⁵I-[Leu⁸,D- Trp²²,Tyr²⁴]SRIF-28 to the various cloned SRIF receptors are shown in the following table wherein the IC₅₀ values are given in nanomolar concentration.

TABLE 1

I I

Peptide No. 3, cyclic H-Cys-Phe-DAgl(NMe,2Np)-IAmp-Tyr-Cys-NH₂ selectively binds to SSTRl with an average IC₅₀ = 6.0 nM, compared to an IC₅₀ >1K nM for SSTR2 and SSTR5, an IC₅₀ of 317 nM for SSTR3 and an IC₅₀ of 481 nM for SSTR4. This hexapeptide containing only 6 residues is a much smaller and appealing molecule than prior SSTRl -selective peptides. The substitution of Cys with D-Cys has little effect on SSTRl binding affinity and function, as shown by testing results for Peptides Nos. 9 and 10.

Peptide No. 6 shows that the addition of a residue at the C-terminus and the N- terminus is tolerated. It exhibits a binding IC₅Q of 0.19 nM and 5, 000-fold selectivity versus SSTR2 and SSTR5, 500-fold selectivity versus SSTR3, and 100-fold selectivity versus SSTR4; it has an EC_5O value of 0.37 nM in the luciferase reporter gene assay.

When Peptide No. 6 was radio-iodinated with ¹²⁵I, it was found to still bind to sections of membrane pellet of SSTRl -expressing transfected cells with high specificity. Moreover, this radioligand was able to label virtually all tested SSTRl -expressing tumors, including a selection of well characterized SSTRl -expressing prostate cancers, mesenchymal cancers, bronchial carcinoids and gastroenteropancreatic tumors.

The peptides of the invention not only provide more selective ligands for binding SSTRl but the use of labeled peptides, for example, a radioiodinated version of one of Peptide Nos. 3, 6 and 10, facilitates drug screening for inhibitors for the receptor, e.g. antagonists that are more effective than those presently known. Competitive binding assays with candidate compounds would first be carried out in this manner with SSTRl to search for high binding affinity; then by screening the multiple SRIF receptors, it could be confirmed whether there was selective binding to only this receptor, as is desired.

Because, as shown above, additions to the N-terminus of the SRIF analog do not adversely affect the selective binding, it should be clear that these compounds can be complexed with a cytotoxic or a radioactive agent for the purpose of carrying that agent to a tumor or other tissue for which degradation is desired. For example, a dialdehyde linker such as glutaraldehyde may be used to link the SRIF analog to saporin or gelonin. Likewise, linkers such as DOTA or DTPA or other suitable chelating agents can be used to complex the SRIF analog with a highly radioactive element as indicated hereinbefore. If desired, the solubility of the SRIF analogs can be improved by acylation of the N-terminal amino group using a hydrophilic compound, such as hydroorotic acid or the like, or by reaction with a suitable isocyanate, such as methylisocyanate or isopropylisocyanate, to create a urea moiety at the N-terminus. Other agents can also be N-terminally linked that will increase the duration of action of the SRIF analog as known in this art.

These SRIF analogs or nontoxic salts thereof, combined with a pharmaceutically or veterinarily acceptable carrier to form a pharmaceutical composition, may be administered to animals, including humans and other mammals, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g. intranasally, intracerebrospinally or orally. The peptides should be at least about 90% pure and preferably should have a purity of at least about 98%; however, lower purities are effective and may well be used with mammals other than humans. This purity means that the intended peptide constitutes the stated weight % of all like peptides and peptide fragments present. Administration to humans should be under the direction of a physician to combat specific tumors and cancers or to mediate other conditions where the SSTRl receptors exert a control function, such as coupling to a tyrosine phosphatase so that stimulation of this enzyme can be carried out to mediate the anti-proliferative effects of SRIF. The required dosage will vary with the particular condition being treated, with the severity of the condition and with the duration of desired treatment.

Such peptides are often administered in the form of pharmaceutically or veterinarily acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like. Illustrative of such nontoxic salts are hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

It may also be desirable to deliver these SRIF analogs over prolonged periods of time, for example, for periods of one week to one year from a single administration, and slow release, depot or implant dosage forms may be utilized as well known in this art. For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of solubility in body fluids, for example, an acid addition salt with a polybasic acid; a salt with a polyvalent metal cation; or combination of the two salts. A relatively insoluble salt may also be formulated in a gel, for example, an aluminum stearate gel. A suitable, slow-release depot formulation for injection may also contain an SRIF analog or a salt thereof dispersed or encapsulated in a slow degrading, non-toxic or non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example, as described in U.S. Pat. No. 3,773,919.

Therapeutically effective amounts of the peptides should be administered under the guidance of a physician, and pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically or veterinarily-acceptable carrier. The SRIF analogs of the invention are generally effective at levels of less than 100 micrograms per kilogram of body weight. A therapeutically effective amount is considered to be a predetermined amount calculated to achieve the desired effect. The required dosage will vary with the particular treatment and with the duration of desired treatment. Generally dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used; however, for prolonged action, it may be desirable to use dosage levels of about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are soluble in water and thus can be prepared as relatively concentrated solutions for administration.

It may be particularly advantageous to administer such compounds in depot or long- lasting form as earlier described. A therapeutically effective amount is typically an amount of an SRIF analog that, when administered peripherally, e.g. intravenously, in a physiologically acceptable composition, is sufficient to achieve a plasma concentration thereof from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1 μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml and usually 5 to 10 μg/ml. In these amounts, they may be used for the prevention of IH, or in appropriate treatments for cardiovascular diseases and other SSTRl -mediated physiopathologies.

Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. Although the claims variously define the invention in terms of a peptide sequence, it should be understood that such is intended to include nontoxic salts thereof which are well known to be the full equivalent thereof and which are most frequently administered.

As previously indicated, these specified modifications can be incorporated in previously disclosed SRIF analogs to create SSTRl -selectivity. The inclusion of certain residues at the N-terminus is optional, but except for Tyr or D-Tyr, such elongation is not considered worthwhile unless it would favorably influence solubility and/or resistance to aminopeptidases. As mentioned, a conjugating agent may be linked to the -amino group of the residue at the N-terminus of these peptides. The inclusion of a residue, such as Thr or NaI at the C-terminus of the 6-member cyclic SRIF analog should not detract from selectivity and may increase the binding affinity to SSTRl. All temperatures are ⁰C and all ratios are by volume. Percentages of liquid materials are also by volume.

Claims

WHAT IS CLAIMED IS:

1. A cyclic somatostatin (SRIF) analog peptide agonist which selectively binds the SRIF receptor SSTRl, said peptide comprising the cyclic amino acid sequence: Cys-Phe(X)-D-Agl(NMe,2Np)-IAmp-Xaa-Cys, where X is H, 4Cl, 4F, 4NO_{2 ,} 4NH₂, 4NH- CONH₂, 4NHCONHOCH₃, 4NHCONHOCH₂-CH₃ or 4NHCONHOH, where Xaa is Phe, Tyr or iodotyrosine (ITyr), where the C-terminus is amidated, where D-AgI(Me, 2Np) stands for D-N^Me,2-naphthoyl aminoglycine, and where IAmp stands for 4-(N-isopropyl)- aminomethylpheylalanine.

2. The peptide of claim 1 wherein Xaa is Tyr.

3. The peptide of claim 2 wherein Thr-NH₂ or 2NaI-NH₂ is present at the C- terminus.

4. The peptide of claim 1 wherein X is 4ClPhe or 4FPhe.

5. The peptide of claim 1 wherein Xaa is Phe.

6. The peptide of claim 1 wherein Xaa is ITyr and I is radioactive iodine.

7. A pharmaceutical composition comprising a mixture of the peptide according to claim 6 and at least one pharmaceutically acceptable carrier.

8. A method for detecting the presence of cells having SSTRl by administering an effective amount of the pharmaceutical composition according to claim 7 so as to selectively bind to such cells and provide a detectable signal at the location thereof and then monitoring for such signal.

9. The peptide of claim 1 wherein a conjugating/complexing agent capable of linking to a cytotoxin or complexing to a radioactive nuclide is present at the N-terminus.

10. The peptide of claim 1 wherein a polyaminopolycarboxylic conjugating agent is present at the N-terminus.

11. The peptide of claim 1 wherein DOTA, DTPA, HYNIC or P₂S₂-COOH is present at the N-terminus.

12. A pharmaceutical composition comprising a mixture of the peptide according to claim 11 and at least one pharmaceutically acceptable carrier.

13. A method for destroying SSTRl -containing cells, which method comprises administering thereto an amount of the pharmaceutical composition according to claim 12 which includes a radioactive nuclide or a cytotoxin, which amount is effective to destroy such cells.

14. A pharmaceutical composition comprising a mixture of the peptide according to any one of claims 1-5, 9 and 10 and at least one pharmaceutically acceptable carrier.

15. A method of treating an SSTRl-mediated physiopathology, which method comprises administering an amount of the pharmaceutical composition according to claim 14, which amount is effective to reach tissue affected thereby which has SSTRl receptors and to activate said receptors.